CN115348870B

CN115348870B - Universal chimeric antigen receptor T cells and uses thereof

Info

Publication number: CN115348870B
Application number: CN202280003210.9A
Authority: CN
Inventors: 胡广; 张佳元; 高文静; 董文洁; 姚小敏; 刘智; 王晓倩
Original assignee: Shanghai Reindeer Biotechnology Co ltd
Current assignee: Shanghai Reindeer Biotechnology Co ltd
Priority date: 2021-03-16
Filing date: 2022-03-16
Publication date: 2023-06-13
Anticipated expiration: 2042-03-16
Also published as: WO2022194224A1; EP4309669A1; CN115348870A

Abstract

A cell comprising a mutant protein which renders the cell insensitive to inhibitors affecting its activity and/or killing function. Universal chimeric antigen receptor T cells associated with said cells suitable for administration to unspecified patients, as well as methods for the preparation of these cells and their use in cell therapy.

Description

Universal chimeric antigen receptor T cells and uses thereof

Technical Field

The present disclosure relates to chimeric antigen receptor T cells, particularly universal chimeric antigen receptor T cells suitable for administration to unspecified patients. The disclosure also relates to methods of making and using these chimeric antigen receptor T cells.

Background

Adoptive immune cell therapy has shown great potential in the treatment of cancer, autoimmune diseases, and infectious diseases. In recent years, cancer therapy using T cells expressing chimeric antigen receptors (CAR-T cells) has been of particular interest. Currently CAR-T cells are mainly prepared using T cells of the patient themselves. This involves isolation, modification and expansion of T cells for each patient, which is time consuming and costly. Furthermore, for neonatal, elderly, etc. patients it is often difficult to obtain T cells with good quality to produce the specific CAR-T cells required for the patient.

One possible solution is to use T cells derived from healthy donors to generate universal CAR-T cells. Compared with autologous CAR-T cells, the universal CAR-T cells have various advantages:

(1) By obtaining a stable batch of frozen cell products, the universal CAR-T product can greatly shorten the waiting period for treatment of patients and provide timely treatment for patients;

(2) Providing a standardized preparation flow for the CAR-T cell product;

(3) There is sufficient time for various modifications to the cells;

(4) Repeated administration can be realized;

(5) Can be combined with CAR-T products with different targets;

(6) Reduces the preparation cost of the CAR-T through an industrialized production flow, and the like.

Thus, universal CAR-T products would be a major trend in future CAR-T therapies.

However, the development of universal CAR-T also faces significant challenges, most of which need to be addressed:

(1) Graft versus host disease GvHD due to allogeneic cell infusion;

(2) The universal CAR-T is rapidly cleared in the host by the host immune system and cannot be amplified efficiently.

The first problem has been solved substantially at present. Researchers knock out TRAC genes encoding T cell surface receptors (TCRs) on alpha beta-T cells through gene editing technology, and effectively inhibit the non-differential attack of the CAR-T cells on host cells by activating the TCRs, thereby avoiding GvHD.

In contrast, the second problem is more difficult to solve. In recent years researchers have been exploring how to effectively expand universal CAR-T cells in a host. Currently, there are two main solutions:

(1) The combination of CD52 mab drug and universal CAR-T. After the application of the monoclonal antibody drug Alemtuzumab of CD52 protein and the chemotherapeutic drug to clear the stranguria, patients were infused with TRAC/CD52 double knocked-out CAR-T cells for treatment. In the scheme, TRAC is knocked out to prevent graft-versus-host disease, CD52 is knocked out to prevent clearance of general CAR-T by stranguria-clearing medicines. Currently, the global disclosure of general CAR-T clinical data is a therapeutic strategy employing a combination of TRAC/CD52 knockout and CD52 mab drugs.

(2) Another strategy to reduce host rejection graft response is to knock out the B2M gene encoding the beta 2-microglobulin on the universal CAR-T. Disruption of the beta 2-microglobulin (B2M knockout) prevents expression of functional HLA-class I molecules on the surface of CAR-T cells. The scheme avoids the general CAR-T from activating cytotoxic T cells in a host body by destroying HLA-I molecules, thereby enabling the host to proliferate for a long time. However, since HLA is an inhibitory ligand for NK cells, its absence activates the clearance of CAR-T cells by patient NK cells, limiting their expansion in vivo, affecting their effectiveness.

To date, researchers have optimized TRAC/B2M knockdown CAR-T cells in a variety of ways, but there is still a high likelihood of immune clearance by the host.

Disclosure of Invention

In one aspect, provided herein are cells comprising mutant proteins that render the cells insensitive to inhibitors that affect their activity and/or killing function.

In some embodiments, the mutant protein belongs to a tyrosine kinase family member.

In some embodiments, the mutant protein is LCK.

In some embodiments, the LCK protein comprises a T316 mutation.

In some embodiments, the LCK protein comprises a T316I, T316A or T316M mutation.

In some embodiments, the inhibitor is a tyrosine kinase inhibitor.

In some embodiments, the inhibitor is dasatinib and/or panatinib.

In some embodiments, the cell is a mammalian cell.

In some embodiments, the cell is a stem cell or an immune cell.

In some embodiments, the immune cell is an NK cell or a T cell.

In some embodiments, the cell expresses a Chimeric Antigen Receptor (CAR).

In some embodiments, the mutant protein is formed by base editing, HDR, and/or over-expression.

In some embodiments, the overexpression is formed by lentivirus, retrovirus, adeno-associated virus, and adenovirus transfection.

In another aspect, provided herein is a method of introducing a T316 mutation in the Lck gene of a cell, comprising introducing a base editor into the cell.

In some embodiments, the base editor is an ABE or CBE base editor.

In some embodiments, the method further comprises introducing sgrnas into the cells.

In some embodiments, the sgrnas comprise SEQ ID NOs: 5-8.

In some embodiments, the CBE base editor is an A3A-CBE3 fusion protein.

In some embodiments, the cell is a mammalian cell.

In some embodiments, the cell is a stem cell or an immune cell.

In some embodiments, the immune cell is an NK cell or a T cell.

In some embodiments, the cell expresses a Chimeric Antigen Receptor (CAR).

In another aspect, provided herein are cells expressing a chimeric antigen receptor, wherein the cells have or are induced to have a killing activity, and the cells are engineered such that their killing activity is insensitive to inhibitors of cellular activity.

In some embodiments, the inhibitor of cellular activity is an inhibitor of T cell activity.

In some embodiments, at least one bioactive molecule in the cell is engineered to be insensitive to the inhibitor of cellular activity, whereas the bioactive molecule is capable of being inhibited by the inhibitor of cellular activity in normal T cells.

In some embodiments, the bioactive molecule functions on a chimeric antigen receptor signaling pathway.

In some embodiments, the biologically active molecule is a protease.

In some embodiments, the protease is a protein tyrosine kinase.

In some embodiments, the protease is an LCK protein.

In some embodiments, the Lck protein tyrosine kinase has a T316 mutation.

In some embodiments, the Lck protein tyrosine kinase has a T316I, T316A or T316M mutation.

In some embodiments, the engineering is accomplished using a base editor.

In some embodiments, the base editor is an ABE or CBE base editor.

In some embodiments, the T316I mutation is obtained by: introducing into said cell a base editor CBE and an sgRNA whose target sequence comprises the nucleotide sequence of SEQ ID NOs: 5-8.

In some embodiments, the LCK protein in the cell comprises SEQ ID NOs: 9-11.

In some embodiments, the cell is a T cell or NK cell.

In some embodiments, the cells are also engineered to eliminate or attenuate cell killing activity generated via their cell surface TCRs.

In some embodiments, the TCR-associated gene of the cell is knocked out.

In some embodiments, the TRAC gene of the cell is knocked out.

In some embodiments, the β2m gene of the cell is knocked out.

In some embodiments, the CIITA gene of the cell is knocked out.

In some embodiments, the T cell activity inhibitor is a protein tyrosine kinase inhibitor.

In some embodiments, the T cell activity inhibitor is an LCK protein tyrosine kinase inhibitor.

In some embodiments, the T cell activity inhibitor is dasatinib and/or panatinib.

In another aspect, provided herein is the use of the above cell in the preparation of a universal CAR-T cell.

In another aspect, provided herein are methods of making a CAR cell comprising engineering the CAR cell such that its CAR-mediated killing activity is insensitive to an inhibitor of T cell activity.

In some embodiments, at least one bioactive molecule of the CAR cell is engineered to be insensitive to the T cell inhibitor, while the bioactive molecule is capable of being inhibited by the T cell activity inhibitor in normal T cells.

In some embodiments, the bioactive molecule acts on the signal transduction pathway of the CAR.

In some embodiments, the biologically active molecule is a protease.

In some embodiments, the protease is a protein tyrosine kinase.

In some embodiments, the enzyme is an LCK protein tyrosine kinase.

In some embodiments, the LCK protein tyrosine kinase has a T316 mutation.

In some embodiments, the LCK protein tyrosine kinase has a T316I mutation.

In some embodiments, the T316I mutation is obtained by: introducing into the CAR cell a cytosine base editor and a sgRNA comprising the amino acid sequence of SEQ ID NOs: 5-8.

In some embodiments, the LCK protein tyrosine kinase in the CAR cell comprises SEQ ID NOs: 9-11.

In some embodiments, the CAR cell is a T cell or NK cell.

In some embodiments, the CAR cell is also engineered to eliminate or attenuate cell killing activity generated via its cell surface TCR.

In some embodiments, the TCR-associated gene of the CAR cell is knocked out.

In some embodiments, the TRAC gene of the CAR cell is knocked out.

In some embodiments, the β2m gene of the CAR cell is knocked out.

In some embodiments, the CIITA gene of the CAR cell is knocked out.

In some embodiments, the T cell or NK cell is contacted with the T cell activity inhibitor during the preparation of the CAR cell from the T cell or NK cell.

In some embodiments, the T cell or NK cell is contacted with the T cell activity inhibitor while the T316I mutation is performed; preferably, the concentration of the T cell activity inhibitor is 100nM.

In some embodiments, the intracellular signaling domain of the CAR comprises:

1) A signaling domain from a CD3z molecule and a co-stimulatory domain from a CD28 molecule; and

2) Optionally, i) hIL7 and CCL19, or ii) IL2RB and IL7Ra variants,

wherein, preferably, the signaling domain from a CD3z molecule comprises SEQ ID NO: 48; the co-stimulatory domain from the CD28 molecule comprises SEQ ID NO:46, an amino acid sequence shown in seq id no; the hIL7 comprises the sequence of SEQ ID NO: 79; the CCL19 comprises the amino acid sequence of SEQ ID NO:80, an amino acid sequence shown in seq id no; the IL2RB and the co-stimulatory domain from the CD28 molecule comprise an IL2RB-CD3z peptide fragment, said IL2RB-CD3z peptide fragment comprising the amino acid sequence of SEQ ID NO:81, and a sequence of amino acids shown in seq id no; the IL7Ra variable comprises SEQ ID NO: 82.

In another aspect, provided herein is a method of treating a disease in a patient comprising administering to the patient an inhibitor of cell activity as described above in combination with a cell.

In some embodiments, the cells are not derived from the patient.

In some embodiments, the cell is a T cell.

In some embodiments, the cytostatic agent is an inhibitor of T cell activity.

In some embodiments, the inhibitor of cellular activity is a protein tyrosine kinase inhibitor.

In some embodiments, the inhibitor of cellular activity is an LCK protein inhibitor.

In some embodiments, the disease is a tumor.

In some embodiments, the method further comprises stopping the administration of the inhibitor of cellular activity, such that the patient's own T cells are restored in activity and the administered cells are cleared; or increasing the amount of the inhibitor of cellular activity administered to inhibit the killing activity of the cells in the patient.

In another aspect, provided herein is a pharmaceutical kit or pharmaceutical combination comprising the above-described cell and an inhibitor of cellular activity.

In some embodiments, the cell is a T cell.

In some embodiments, the cytostatic agent is a protein tyrosine kinase inhibitor.

In another aspect, provided herein is the use of the above cell in combination with an inhibitor of cellular activity in the preparation of an anti-neoplastic medicament.

In some embodiments, the cell is a T cell.

The cells provided herein, and the universal CAR-T cells made therefrom, can be used to treat non-specific patients (allogeneic cell therapy), overcoming a series of problems associated with the need for existing CAR-T cells to be derived from the patient itself.

Drawings

FIG. 1 is a schematic diagram of UCART strategy principle in the case of dasatinib effectively inhibiting host T cells.

FIG. 2 is a schematic diagram of UCART strategy principle in the case of dasatinib effectively inhibiting host NK cells.

FIG. 3 is a schematic representation of LCK-mediated TCR signaling.

FIG. 4 is a schematic representation of the binding relationship of dasatinib to different LCK mutations.

FIG. 5 shows a schematic representation of conservation of ABL and LCK kinase and base changes of LCK mutations.

FIG. 6 is a graph showing the design and distribution of LCK-T316 mutation-related sgRNA.

FIG. 7 shows the prediction of off-target and efficiency of LCK-T316 mutation-related sgRNA.

FIG. 8 is a schematic diagram of sgRNA design and distribution suitable for CBE3 induced LCK-T316I mutation.

FIG. 9 shows that LCK-T316I is capable of tolerating anti-BCMA CAR-T cells to inhibition of cell proliferation by dasatinib.

FIG. 10 shows single base editing for human LCK ^T316I Schematic representation of the design of mutated sgrnas and specific sequences. 10A induction of human LCK ^T316I Schematic representation of mutant sgrnas design. The first row of numbers indicates the amino acid sequence numbers of the LCK coding region and the second row indicates the type of amino acid. Capital letters are exon regions and lowercase letters are intron regions in the third row of original gene sequences. The fourth and fifth row illustrate the targeting sequence of the sgrnas, i.e. the sgRNA sequences and PAM sequences not marked by the black arrows, respectively, coinciding with the black arrows. The seventh row in the sixth row represents the gene sequence and amino acid sequence, respectively, after the desired mutation. The red color represents the mutation of interest. 10B. Specific sequence of the designed sgRNA. For LCK-sgRNA12 and LCK-sgRNA16 minAnd (3) carrying out length optimization design, namely carrying out specific length and sequence, namely PAM and other information of 7 sgRNAs which are designed in a total range from 18nt to 21 nt.

FIG. 11 shows that LCK-T316I mutations can be induced efficiently by LCK-sgRNA16 of different lengths. 11A. Electrotransfer editing of T cells after 96hr, sequencing of LCK-T316 site peak map results. LCK-sgRNA16 of 18nt, 20nt and 21nt lengths had about 50% mutation induction at cytosine at the T316 site. 11B. Specific analysis results of mutation efficiency using the EditR for the peak pattern in 11A.

FIG. 12 shows that LCK-T316I mutations cannot be induced by LCK-sgRNA12 of different lengths. 12A. Electrotransfer editing of T cells after 96hr, sequencing of LCK-T316 site peak map results. Cytosine at the T316 site in LCK-sgRNA12 of 18nt, 20nt and 21nt lengths did not induce efficient mutation. 12B. Specific analysis results of mutation efficiency using the EditR versus the peak pattern in 12A.

Fig. 13 shows that dasatinib is effective in inhibiting TCR signaling to inhibit T cell activation. T cells were treated with DMSO, 100nM and 1000nM dasatinib for 5 hours, followed by activation or deactivation with anti-CD 3/CD28 DynaBeads for 24 hours, and expression of CD25, CD69 (13A) and 4-1BB (13B) was detected in a flow assay.

FIG. 14 shows inhibition of TCR activation by LCK-T316I mutation to render T cells resistant to dasatinib. 14A,14B. The flow schematic shows the CD25, CD69 (14A) and 4-1BB (14B) expression of T cells under different processing and editing conditions. The first row was devoid of dasatinib and anti-CD 3/CD28 stimulation treatment, the second row was devoid of dasatinib but added anti-CD 3/CD28 stimulation treatment, and the third row was pretreated with 100nM dasatinib for 7 hours followed by anti-CD 3/CD28 stimulation treatment. MockT is a non-electrotransport control, group 01 is an electrotransport-only CBE3 protein control, groups 02-05 are LCK-free synonymous mutation controls edited by CBE3 and sgRNA12, and groups 06-08 are LCK-T316I mutation groups edited by CBE3 and sgRNA 16. 14C. statistics of differences in the expression ratios of T cell activation markers from different groups, based on 14A,14B data.

FIG. 15 shows that LCK-T316I renders anti-CD 19-CAR-T cells resistant to inhibition of CD107a transport by dasatinib. 15A. Sequencing results to detect LCK editing efficiency after 72 hours of anti-CD 19-CAR-T cell electrotransformation and EditR analysis results. Only the LCK-sgRNA16 edited group was able to cause LCK-T316I mutation with a mutation efficiency of 43%.15B. Experimental procedure thumbnail image of example 5, pretreatment of CAR-T cells with 100nM dasatinib for 12 hours, activation of CAR-T cells with target cells, and detection of CD107a release after 5 hours. 15℃ Gate-on strategy for flow results analysis, CD107a release of CD8 and CAR double positive T cells was analyzed. 15D, flow result analysis. The left column shows CD107a release of CAR-T cells incubated with negative target cell K562 in DMSO and 100nM dasatinib, and the right column shows CD107a release of CAR-T cells incubated with positive target cell Nalm6 in DMSO and 100nM dasatinib. Three rows of T cells were the CBE3 electrotransfer control, LCK-sgRNA12 and LCK-sgRNA16 edited groups, respectively. 15E. FIG. 15D statistical analysis of the streaming results.

FIG. 16 shows LCK-T316I tolerates anti-BCMA-CAR-T cells with inhibition of CD107a transport by dasatinib. Sequencing results to detect LCK editing efficiency after 72 hours of anti-BCMA-CAR-T cell electrotransformation and edit analysis results. Only the LCK-sgRNA16 edited group was able to cause LCK-T316I mutation with a mutation efficiency of 42%. And 16B, flow type result analysis. The left column shows CD107a release of CAR-T incubated with negative target cell K562 in DMSO and 100nM dasatinib, the middle column shows CD107a release of CAR-T incubated with positive target cell U266B1 in DMSO and 100nM dasatinib, and the right column shows CD107a release of CAR-T incubated with positive target cell RPMI8226 in DMSO and 100nM dasatinib. Three rows of T cells were the CBE3 electrotransfer control, LCK-sgRNA12 and LCK-sgRNA16 edited groups, respectively. Fig. 16℃ Statistical analysis of the flow results of fig. 16B.

FIG. 17 shows LCK ^T316I Mutations render CD19/BCMA bispecific CAR-T cells resistant to inhibition of CD107a transport by dasatinib. 17a.cd107a streaming results statistics. Each row represents the release of CD107a by co-incubation of different types of target cells with DMSO or 100nM dasatinib treatment of CD19/BCMA bispecific CAR-T of different edit types. 17B. bar graph display of CD107A release ratio in FIG. 17A.

Figure 18 shows that LCK-T316I mutation renders anti-BCMA-CAR-T cells tolerant to inhibition of CAR-T killing function by dasatinib. The experimental procedure of example 8 is outlined in that CAR-T cells are pretreated with 100nM dasatinib for 12 hours and then after co-culturing the target cells with CAR-T cells for 24 hours, the activity of the luciferase of the target cells is detected to indicate the killing of the target cells by CAR-T. And 18B, detecting the luciferase activity value. The leftmost column is the luciferase basal values for only CAR-T cells without target cells, and the middle and rightmost columns are data for CAR-T cells under different treatments, co-incubated with negative target cells Nalm6 and with positive target cells RPMI-8226, respectively. Histogram sort and significance analysis results were performed on the data in fig. 18B.

Figure 19 shows that LCK-T316I mutation renders anti-BCMA-CAR-T cells resistant to inhibition of CAR-T killing by dasatinib (repeated experiments). 19A, luciferase activity value detection result. The leftmost column is the luciferase basal values for only CAR-T cells without target cells, and the second column to the fourth column are data for CAR-T cells co-incubated with negative target cells K562, raji, and positive target cells RPMI-8226, respectively, under different treatments. 19B. histogram sort and significance analysis results were performed on the data in FIG. 19A.

Figure 20 shows that dasatinib at a concentration of 25nM is effective in exerting an inhibition of T cell activation function in vitro. Flow assay expression profiles for T cells CD25 and CD69 under different treatments. 2 duplicate wells were treated each. 20 B.T cell CD25 and 4-1BB flow assay expression patterns under different treatments. 2 duplicate wells were treated each.

Figure 21 shows that there is a continued effect of dasatinib on the inhibition of T cell activation function. Concentration experiments were adjusted after 21a.50nm dasatinib pretreatment. On the left is the CD107a release result of the streaming assay. The first row is Sg12 edited LCK synonymous mutant CD5-UCAR-T cells, and the second row is Sg16 edited LCK-T316I mutated CD5-UCAR-T cells. The dasatinib concentration was reduced from the leftmost column to the rightmost column to 0, 15, 25, 35 and 50nM, respectively. The right hand bar graph shows CD107a positive rate. Concentration experiments were adjusted after 21b.25nm dasatinib pretreatment. On the left is the CD107a release result of the streaming assay. The first row is Sg12 edited LCK synonymous mutant CD5-UCAR-T cells, and the second row is Sg16 edited LCK-T316I mutated CD5-UCAR-T cells. The dasatinib concentration was reduced from the leftmost column to the rightmost column to 0, 10, 15, 20 and 25nM, respectively. The right hand bar graph shows CD107a positive rate.

Figure 22 shows that there is a continued effect of dasatinib on T cell activation function inhibition (repeated experiments). 22a.50nm dasatinib pretreatment followed by concentration adjustment, CD107a release results of the flow assay. The leftmost column is a positive control group untreated with dasatinib, decreasing dasatinib concentrations from the second to the right most column to 0, 10, 20, 30, 40 and 50nM, respectively. 22b.25nm dasatinib pretreatment followed by concentration adjustment, CD107a release results of the flow assay. The leftmost column is a positive control group untreated with dasatinib, decreasing dasatinib concentration from the second column to the right column to 0, 10, 20, 25nM, respectively.

FIG. 23 shows that 100nM dasatinib is effective in inhibiting CD107a transport during NK cell activation. Results of CD107a release of NK cells by flow assay. The first row of NK cells was treated with different concentrations of dasatinib without K562 activation. The second row of cells was treated with different concentrations of dasatinib with K562 addition for activation.

FIG. 24 shows that dasatinib at a concentration of 30nM is effective in exerting an inhibition of NK cell activation function in vitro. 24A.0-50nM dasatinib gradient treatment on NK cell CD107a release flow chart. The concentrations from the leftmost column in the upper row to the rightmost column in the lower row were 0, 10, 20, 25, 30, 40 and 50nM, respectively. 24B histogram results of CD107a positive rate in FIG. 24A.

Figure 25 shows that there is a continued effect of dasatinib in vitro on the inhibition of NK cell activation function. Concentration was adjusted after pretreatment of dasatinib at different concentrations (0, 25 and 50 nM), NK cell CD107a release results of flow assay. The dasatinib concentration was reduced from the leftmost to the rightmost group to 0, 7.5, 12.5, 17.5 and 25nM, respectively. Bar graph of CD107a positive rate in fig. 25A. The results show that there is a continued effect of dasatinib on the inhibition of NK cell activation function.

Figure 26 shows that there is a continued effect of dasatinib in vitro on NK cell activation function inhibition (repeated experiments). 26a.50nm of dasatinib was pre-treated and the concentration was adjusted and NK cells CD107a was flow tested for release results. The leftmost column is a positive control without any treatment, decreasing dasatinib concentrations from the second left column to the rightmost column to 0, 10, 20, 30, 40 and 50nM, respectively. 26b.10nM and 25nM of dasatinib were pre-treated and the concentration was adjusted and NK cell CD107a release results were detected by flow. The leftmost column is a positive control without any treatment, decreasing the concentration from the left two and three columns to 0 and 10Nm, respectively, and decreasing the dasatinib concentration from the left fourth column to the rightmost column to 0, 10, 20 and 25Nm, respectively.

FIG. 27 is a schematic diagram showing the measurement of the parameters for the preparation of CD 5-UCART. 27A. TRAC gene knockout efficiency was 99.8% according to flow cytometry analysis; the knockout efficiency of the CD5 gene was 99.9%. About 35% of CAR expressing T cells were measured by detection of EGFR transduction markers with CD5 antigen. 27C. CBE3 group is CD5-UCAT cell control group, sg12 group is CD5-UCAT cell control group with synonymous mutation to LCK, neither control group has LCK ^T316I Mutation; the sg16 group (Km group) was the occurrence of LCK ^T316I The editing efficiency was 79%.

FIG. 28 shows that LCK-T316I mutated CD5-UCAR-T cells have an expansion advantage in MLR reactions mixed with allogeneic T. Schematic of the ratio of CD5-UCAR-T cells in whole cells at various time points in the MLR reaction. Schematic of the ratio of allogeneic T cells to whole cells at various time points in the mlr response. Schematic of the change in CAR positive rate at different time points in mlr reaction. 28D. UCART to allogeneic T cell ratio variation at various time points in the MLR reaction.

FIG. 29 is a schematic diagram showing the measurement of the parameters for the preparation of CD 5-UCART. 29A. TRAC, CD5 knock-out efficiency assay. 29 b.5 days after lentivirus infection of t cells, CAR positive rate results were detected by flow cytometry with anti-EGFR antibodies. 29C. efficiency detection of Km editing by CD5-UCART cells: km editing did not occur in the control group; the mutation efficiency of T316I in the sg16 (Km) group CD5-UCART cells was 45%.

FIG. 30 shows that LCK-T316I mutated CD5-UCAR-T cells have an expansion advantage in MLR reactions mixed with allogeneic T. Schematic of total CD5-UCAR-T cells at various time points in the MLR reaction. Schematic of the ratio of CD5-UCAR-T cells in whole cells at various time points in the MLR reaction. Schematic of total allogeneic T (mock host T) cells at various time points in the mlr response. Schematic of the ratio of allogeneic T (mock host T) cells to whole cells at various time points in the mlr response. Schematic of the change in the proportion of UCART cells expressing CAR molecules at different time points in the mlr reaction. The ratio of UCART to mock host T cells at various time points in the 30f.mlr reaction (real-time effective target ratio) is schematically varied.

FIG. 31 shows inhibition of T cell function by Km-edited UCAR-T cells tolerating dasatinib. LCK gene mutation sequencing results for all cells in the 11 day system of mixed lymphoresponse. 31B. bar graph significance analysis results of FIG. 31A.

FIG. 32 shows the results of expression analysis of surface marker molecules at concentrations of 0-200nM of panatinib. Sanger sequencing examined the mutation efficiency of the LCK-T316I region of three different editing-type T cells. The Sg16-Km group had a mutation of 30%, whereas the control group had substantially no mutation. 32B. Experimental procedure thumbnail. Panatinib treatment was performed at different concentrations on T cells of different edit types, with panatinib-free left-most column and no control group activated. The panatinib pretreatment was performed at 0, 10, 50, 100 and 200nM from the second column to the rightmost column, respectively, and the active group was added. The APC-labeled murine IgG monoclonal antibody detects activated T cell surface marker molecule 4-1BB, and the PE-labeled murine IgG monoclonal antibody detects activated T cell surface marker molecule CD25. Experiments were performed with 32C, FITC-labeled murine IgG monoclonal antibody to detect activated T cell surface molecule CD69, and PE-labeled murine IgG monoclonal antibody to detect activated T cell surface molecule CD25.

FIG. 33 shows the results of expression analysis of surface marker molecules at concentrations of 0-1000nM of panatinib. Panatinib treatment was performed at different concentrations on T cells of different edit types, with panatinib-free left-most column and no control group activated. The second column to the rightmost column were pretreated with 0, 10, 50, 200, 500 and 1000nM panatinib, respectively, and the active group was added. The APC-labeled murine IgG monoclonal antibody detects activated T cell surface marker molecule 4-1BB, and the PE-labeled murine IgG monoclonal antibody detects activated T cell surface marker molecule CD25. 33B. Experiments were performed with 23C, FITC-labeled murine IgG monoclonal antibody detecting activated T cell surface molecule CD69, and PE-labeled murine IgG monoclonal antibody detecting activated T cell surface molecule CD25.

FIG. 34 shows that panatinib at a concentration of 200nM is effective in exerting an inhibition of NK cell activation function in vitro. CD107a release flowsheet for NK cells by 34A.0-500nM panatinib concentration gradient treatment. The concentrations from the leftmost column in the upper row to the rightmost column in the lower row were 0, 50, 200 and 500nM, respectively. 34B. histogram results of CD107a positive rate in FIG. 34A, 200nM panatinib was able to significantly inhibit NK cell release by K562 activated CD107 a.

Figure 35 shows the continued effect of panatinib on the inhibition of T cell activation function. 35A.0, 50 and 200nM panatinib pretreatment followed by concentration experiments, CD107a release results of the flow assay. The first and second rows are panatinib-free incubation groups. Third and fourth rows were 200nM pretreatment followed by lowering the panatinib concentration to 0, 50, 100 and 200nM. Fifth and sixth rows were 500nM pretreatment followed by lowering the panatinib concentration to 0, 50, 100, 200 and 500nM. Histogram of CD107a positive rate of data in 35A for concentration experiments adjusted after 35b.0, 50 and 200nM panatinib pretreatment. The results show that panatinib has a continuous effect on the inhibition of T cell activation function.

FIG. 36 shows the continued effect of panatinib on NK cell activation function inhibition in vitro. Concentration was adjusted after panatinib pretreatment at different concentrations (0, 50, 100 and 200 nM), NK cell CD107a release results of flow assay. The dasatinib concentration was reduced from the leftmost to the rightmost group to 0, 50, 100 and 200nM, respectively. Fig. 36B bar graph of CD107a positive rate of fig. 36A. The results show that panatinib has a continuous effect on the inhibition of NK cell activation function.

Figure 37 shows the possibility of panatinib as a combination drug and switch for general CAR-T treatment. After 37a.200nM panatinib pretreatment overnight, CD19-UCART at different concentrations (0, 50, 100 and 200 nM) of panatinib treated was incubated with target cells for 4 hours and CD19-UCART cells were assayed for CD107a release. After 37b.500nM of panatinib pretreatment overnight, CD19-UCART at different concentrations (0, 100, 200 and 300 nM) of panatinib treated was incubated with target cells for 4 hours and CD19-UCART cells were assayed for CD107a release.

FIG. 38 shows that LCK-T316I mutated B2M deficient CD19-UCAR-T cells have an expansion advantage in MLR reactions mixed with allogeneic CD3 deleted PBMC. 38A. Schematic of the knockout efficiency of UCART cell B2M, TRAC and CAR positive detection prepared. 38B. line plot of UCART cell total amount over time in vitro mixed lymphocyte reaction experiments. 38C. line graph of CAR positive rate over time in vitro mixed lympho-response experiments. 38D. line plot of CD19 positive cell occupancy versus time in vitro mixed lymphocyte reaction experiments. 38E. Histogram of total CAR positive cells over time at different target ratios in vitro mixed lymphoresponse experiments.

FIG. 39 shows the parameters of CD19-UCART production and the results of the assay. 39a.trac, B2M, CIITA gene knockout efficiency detection and 5 days after cell infection with lentivirus, flow cytometry detection of knockout rate and CAR positive rate results. 39b. Efficiency detection of Km editing by cd19-UCART cells: km editing did not occur in the control group; the mutation efficiency of T316I in the sg16 (Km) group CD19-UCART cells was 20%.

FIG. 40 shows that LCK-T316I mutated CD19-UCAR-T cells have an expansion advantage in MLR reactions mixed with allogeneic PBMC. Schematic of the total amount of CD19-UCAR-T cells at various time points in the MLR reaction. Schematic of the ratio of CD19-UCAR-T cells in whole cells at various time points in the MLR reaction. Schematic of total allogeneic T (mock host T) cells at various time points in the mlr reaction. Schematic of total cell count in whole cells of allogeneic NK (mock host NK) cells at various time points in the mlr response. Schematic of total cell mass expressing CAR molecules at different time points in the mlr reaction. Schematic of the ratio of CAR molecule-expressing cells in whole cells at different time points in the mlr reaction.

FIG. 41 shows the results of the detection of the optimization of the CD19-UCART production process parameters. 41A.TRAC, B2M gene knockout efficiency assay. 41B. 5 days after lentivirus infection of cells, flow cytometry examined CAR positive rate results. 41c. efficiency detection of Km editing by cd19-UCART cells: the mutation efficiency of the experimental group T316I is obviously higher than that of the control group. Eff. Of Km: efficiency of Km mutation.

Fig. 42 shows the construction and results of CARs carrying a third signaling element. 42A. Schematic structural diagram of a plasmid containing CD19-UCART of various third signal structures. 42B. the efficiency of electrokinetic knockdown was shown by detecting TRAC and B2M expression. 42℃ Detection of the CAR+ fraction of UCART cells by scFv binding of CD19-Anti and Anti-CD19 on the CAR structure.

Figure 43 shows the repeated antigen stimulation procedure and results. 43A. Schematic of the flow of the repeated antigen stimulation experiment. 43B-c. CD19-UCART containing various third signal structures in experiment I was used in the effective target ratio of 2 in M1 culture conditions (CTS basal T cell medium does not supplement any cytokines) (43B) and M2 culture conditions (CTS basal T cell medium+200U/ml IL 2) (43C) with Raji cells pretreated with mitomycin C: 1 co-cultivation. The amount of car+ cells per time was obtained from the flow and cell counts to obtain the fold expansion of car+ cells. 43D-E. CD19-UCART containing various third signal structures in experiment II was used in the following ratio of target 2 in M1 culture conditions (CTS basal T cell culture medium does not supplement any cytokines) (43D) and M2 culture conditions (CTS basal T cell culture medium+200U/ml IL 2) (43E) with Raji cells pretreated with mitomycin C: 1 co-cultivation. The amount of car+ cells per time was obtained from the flow and cell counts to obtain the fold expansion of car+ cells.

Fig. 44 shows the functional detection result of CART carrying the third signal structure. 44A. CD19-UCART containing various third signal structures in experiment I residual CD19-UCART after repeated antigen stimulation with Raji-luc cells under M1 culture conditions (CTS basal T cell culture medium does not supplement any cytokines) and M2 culture conditions (CTS basal T cell culture medium +200U/ml IL 2) were measured as 1:1 Co-cultivation the luciferase killing experiment was performed. Luminosity of luciferases after co-cultivation was measured to calculate the proportion of killing Raji-luc and plotted. 44B-c. the remaining CD19-UCART containing various third signal structures after repeated antigen stimulation in M1 culture conditions (CTS basal T cell medium without any cytokine supplementation) and M2 culture conditions (CTS basal T cell medium +200U/ml IL 2) was expressed as 2:1 and 0.5:1 Co-cultivation the luciferase killing experiment was performed. The luminosity of luciferases after co-cultivation was measured, the ratio of killing Raji-luc was calculated and a line graph was drawn. 44D. CD19-UCART containing various third signal structures in experiment II residual CD19-UCART and Raji after repeated antigen stimulation under M1 culture conditions (CTS basal T cell culture medium does not supplement any cytokines) and M2 culture conditions (CTS basal T cell culture medium +200U/ml IL 2) were cultured in a ratio of 1:1 and 0.2:1 Co-culture 107a release assay was performed. Flow results of 107a after co-cultivation were measured to calculate the proportion of UCART cells released 107a and a line graph was drawn.

Fig. 45 shows cytokine secretion results of CART carrying the third signal structure. 45A. CD19-UCART containing various third signal constructs in experiment I the concentration of IL2 was determined from the supernatant of the medium after repeated antigen stimulation under M1 culture conditions (CTS basal T cell medium without any cytokine supplementation) and M2 culture conditions (CTS basal T cell medium+200U/ml IL 2). 45B-D. CD19-UCART containing various third signal constructs in experiment II the concentration of IL2 (45B), IFN-gamma (45C) and TNF-alpha (45D) was determined from the supernatant of the medium after repeated antigen stimulation under M1 culture conditions (CTS basal T cell medium without any cytokine supplementation) and M2 culture conditions (CTS basal T cell medium +200U/ml IL 2).

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

"chimeric antigen receptor (chimeric antigen receptor, CAR)" refers herein to an engineered membrane protein receptor molecule that can confer desired specificity to immune effector cells, such as the ability to bind to a particular tumor antigen. Chimeric antigen receptors are generally composed of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen binding domain is a scFv sequence responsible for recognizing and binding to a specific antigen. Intracellular signaling domains typically include an immune receptor tyrosine activation motif (ITAM), such as a signaling domain derived from a CD3z molecule, responsible for activating immune effector cells and producing killing. In addition, chimeric antigen receptors may also include a signal peptide at the amino terminus that is responsible for intracellular localization of the nascent protein, as well as a hinge region between the antigen binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may also include a costimulatory domain derived from, for example, a 4-1BB or CD28 molecule.

"bispecific chimeric antigen receptor" is intended to mean that the molecule comprises at least two different antigen binding sites in the extracellular antigen binding domain, which recognize and bind respectively different antigen molecules on the target cell. For example, in the examples herein reference is made to bispecific chimeric antigen receptors that target CD19 and BCMA simultaneously.

"CAR cell" refers herein to a cell that expresses a CAR molecule on the surface of a cell. In most cases, the cell is an immune cell, such as a T cell, or NK cell. Accordingly, a T cell expressing a CAR is referred to herein as a "CAR-T" or "CAR-T cell. In addition, when referring to CAR-T cells herein, unless otherwise indicated, reference is made not only to cells directly modified by the CAR, but also to daughter cells produced after proliferation of these cells in vitro or in vivo.

By "universal CAR-T cell (UCAR-T)" is meant herein that such cells are not limited to CAR-T cells that are infused into a particular patient. In the prior art, in order to prevent GvHD and host rejection of the graft, cells (e.g., T cells) are typically harvested from the patient and CAR modified and returned to the patient. Not only is this method time consuming and expensive, but in some cases a sufficient number of patient T cells are not available for CAR modification. In contrast, universal CAR-T cells herein mean that they are suitable for allogeneic transplantation, that the same batch of CAR-T cells can be used for different patients, and that these universal CAR-T cells are not typically derived from these patients.

The present invention is based, at least in part, on the discovery by the inventors of novel general CAR-T preparation methods and use strategies.

In order to prepare universal CAR-T cells (UCAR-T), in our design of universal CAR-T cells, an attempt was made to find a simple and easy to handle protocol that has the following two key points:

(1) Under the scheme, the activation and killing activities of host T cells and NK cells are effectively inhibited, and allogeneic general CAR-T cells are not attacked;

(2) Under the scheme, the functions of activation, killing, amplification and the like of the general CAR-T are not affected.

To inhibit the killing activity of the host T cells and NK cells, inhibitors having inhibitory activity on both or on both, respectively, may be used, for example small molecule inhibitors or inhibitory antibody molecules, which may result in a reduced or no influence on the number of T cells and/or NK cells. In this case, to achieve point (2), the CAR-T cells may be engineered to be insensitive to these inhibitors (in particular such that the CAR signaling pathway is resistant to the inhibitory effects of these inhibitors), i.e. in the presence of these inhibitors, they may still survive, be activated by and have target cell killing activity (even if there is a reduction in killing activity overall). There are a number of ways in which such engineering of CAR-T cells can be performed, including, but not limited to, introducing into the CAR-T cells a genetic mutation, the mutated gene product (e.g., protein or enzyme) being insensitive to these inhibitors and functioning as an unmutated gene product; introducing into the CAR-T cell exogenous genes whose expression in the CAR-T cell produces a protein or enzyme that is unaffected by the inhibitors and whose target molecules that can replace the inhibitors act intracellularly; introducing into the CAR-T cell a conjugate of the inhibitors (e.g., a foreign protein such as an endosome capable of binding to the inhibitors) so as to neutralize the inhibitors; target molecules (e.g., proteases) for these inhibitors are overexpressed in CAR-T cells in order to counteract the effects of these inhibitors, and so forth.

Methods for introducing gene mutations into cells are known in the art and include, but are not limited to, DNA homologous recombination, specific site-directed cleavage with endonucleases (e.g., ZFNs and TALENs), CRISPR-based gene editing techniques, and various base editors (e.g., CBEs, ABEs, and various modified variants thereof, etc.).

Methods of introducing exogenous genes into cells are known in the art and include, but are not limited to, electroporation, gene gun, microinjection, liposome introduction, viral transduction (e.g., using retroviruses, lentiviruses, various modified viral vectors such as adenovirus and adeno-associated viral vectors, etc.).

Methods for allowing a cell to overexpress certain proteins or enzymes may include, for example, using increased copy numbers of genes encoding them, enhancing promoter functions of genes encoding them, and the like.

The introduction of a mutation, exogenous gene, or overexpression into a cell may be short-term or transient, or may be permanent (e.g., integration of the mutated gene or exogenous gene into the host cell chromosome). Introduction may take the form of DNA or RNA, for example, by lentiviral transduction such that the exogenous gene may be expressed in the host cell for a long period of time, or mRNA may be introduced such that the host cell may express some exogenous protein or enzyme for a short period of time.

As used herein, "mutant protein" refers to a protein that has been altered in amino acid sequence relative to wild-type, or that has been altered in expression relative to the level of expression in normal cells, such as increased expression (otherwise known as overexpression).

It will be appreciated by those skilled in the art that the above-described engineering can be performed prior to, simultaneously with, or after modification of the original cell into a CAR-T cell. For example, T cells can be engineered as described above to be insensitive to T cells and NK cell inhibitors, cultured and prepared for T cell libraries, and then CAR modified according to various therapeutic needs to obtain universal CAR-T cells for various therapeutic purposes (e.g., different cancers).

"primordial cells" herein refer to cells of a subject to be or prepared for CAR modification, e.g., stem cells, immune cells at various stages of development, T cells, NK cells, etc. It is contemplated that one of the objects of the present application is to prepare universal CAR-T cells that are independent of the source of the patient to be treated, and thus, the source of these primordial cells may be substantially unrestricted, e.g., from a blood bank, healthy volunteers, etc.

In some embodiments, the inhibitor is a tyrosine kinase inhibitor. In some embodiments, these tyrosine kinases act on the CAR signal transduction pathway. In some embodiments, these tyrosine kinases act simultaneously on the TCR signaling pathway and the CAR signaling pathway. In some embodiments, the tyrosine kinase is an LCK kinase. In some embodiments, the tyrosine kinase is LCK kinase and the inhibitor is Dasatinib (Dasatinib, DS) and/or panatinib (Ponatinib, PN).

In a specific embodiment, the engineering of LCK kinase involves a mutation at its T316 site, e.g., T316I, T M, T a, as well as other mutations, so long as the mutation renders the mutated product insensitive to the inhibitors described above (e.g., dasatinib and panatinib), but at the same time has pre-mutation protease function. Preferably, the mutation is T316I.

In the case of the T316I mutation, the present disclosure provides a smart way to achieve the mutation. The method involves the use of cytosine base editors used in conjunction with sgrnas that effect the conversion of base pairs c.g to t.a on the Lck gene. This base pair transition produces a T316I mutation in its expression product LCK, while the cytosine base editor also causes some other base pair C.G transitions near the mutation site of interest, but these other transitions are just synonymous mutations, not producing amino acid changes in LCK. It will be appreciated by those skilled in the art that although the preferred techniques for creating T316I mutations are described in the specific examples herein, mutations at the T316 site may be created in other similar ways without being excluded. For example, other mutations at the T316 site may be made using gene editing techniques, or specific sequences of the sgRNA may be altered (including length changes, target sequence variations), or even multiple mutations may be made in the product LCK (including the T316 site) using these or other means, as long as the resulting product LCK retains its original function and is insensitive to the inhibitors described above, and such modifications are intended to be included within the scope of the invention.

To prevent the CAR-T cell from attacking the host normal cell (non-target cell), gvHD, it is contemplated that the TCR-related gene on the CAR-T cell may be engineered to have its TCR lose or have its activity reduced, e.g., by gene editing or otherwise knocking out the TRAC gene encoding the TCR receptor alpha chain, and/or the TRBC gene encoding the TCR receptor beta chain.

Further, to prevent the attack of CAR-T cells by a few host T cells or NK cells, it is contemplated to reduce or avoid the expression of HLA-class I molecules on CAR-T molecules, for example by knocking out the B2M gene encoding β2 microglobulin.

The universal CAR-T cells provided herein can be used in combination with an inhibitor of cellular activity for the treatment of a patient (e.g., a cancer patient). As already set forth above, these cell activity inhibitors can be of various classes, provided that they can be in the host (recipient of CAR-T cells) such that their immune system does not result in a loss of target cell killing activity of the infused CAR-T cells. Typically, these inhibitors may be such that the host immune system (e.g., T cells, particularly cd8+ T cells) does not kill the CAR-T cells. In this case, the input CAR-T cells can be activated by the target cells and exert a killing effect, and at the same time, can proliferate in the host body and exert a long-acting effect. Preferably, these inhibitors only inhibit the function of the host immune system without causing disruption of its function. In this case, the host's immune system may resume its original function as soon as possible, either when the inhibitor is cleared or when the inhibitor is not administered further.

Thus, in some embodiments, these inhibitors may be used as molecular switches of whether or not the universal CAR-T cell continues to function. For example, the universal CAR-T cells may be administered to the patient with an inhibitor prior to their administration to the patient to inhibit or attenuate their immune system (particularly T cells) killing activity against the universal CAR-T cells to be administered, followed by administration of the universal CAR-T cells to the patient, and then optionally continued to be administered to the patient periodically with the inhibitor (the same or a different inhibitor than the previous inhibitor) to maintain the concentration of the inhibitor in the patient to a level that continues to inhibit the immune system function of the patient. Because these infused universal CAR-T cells are engineered to be resistant to the inhibitors described above, they recognize and kill their target cells (e.g., cancer cells). After the desired therapeutic effect (e.g., tumor regression or disappearance) is achieved, inhibitor administration is stopped, the patient's autoimmune system is restored and universal CAR-T cells are cleared in vivo. During this process, the level of universal CAR-T content and activity and disease status in the patient can be periodically checked to determine if re-entry of universal CAR-T cells is required. The inhibitors are used as molecular switches, and are also beneficial to the safety of medication. If the patient fails to tolerate universal CAR-T cell therapy, inhibitor use can be stopped at any time to clear these universal CAR-T cells to avoid serious adverse effects. In addition, by increasing the concentration of the inhibitor (e.g., from 200nM to 500nM when panatinib is used as the inhibitor), the universal CAR-T cells described above that can tolerate the inhibitory effect of a dose of inhibitor become unable to tolerate the inhibitor effect and, in turn, unable to continue to proliferate and/or have target cell killing activity. Thus, the killing activity of the universal CAR-T cells in vivo can be controlled by either stopping the use of the inhibitor or increasing the concentration of the inhibitor as a molecular switch.

As set forth in the examples below, in the case of dasatinib as an inhibitor, the dasatinib may be administered in such a dose as to inhibit or attenuate the immune system function of the patient such that the concentration of dasatinib in the patient is not less than 10nM, or not less than 25nM, or not less than 30nM, or not less than 50nM, or not less than 100nM. In the case of panatinib as an inhibitor, it may be administered in such a dose that the concentration of panatinib in the patient is not lower than 100nM, or not lower than 200nM, or not lower than 300nM.

The universal CAR-T cells provided herein can also be used in combination with other cancer therapeutic agents, i.e., administered to a patient with the other cancer therapeutic agents during, before, or after administration of the inhibitor and universal CAR-T cells to the patient. These other cancer therapeutic agents may include chemotherapeutic agents, radiotherapeutic agents, other biologic therapeutic agents such as antibody therapeutic agents or cell therapeutic agents.

As used herein, "patient" refers to a subject and may include any mammal, such as a cat, dog, sheep, cow, mouse, rat, rabbit, human, non-human primate, and the like. In addition, the patient may be any individual who has suffered a disease, is at risk for suffering from a disease, or has been treated. Accordingly, the methods of making and methods of treatment of the universal CAR-T provided herein are useful in human and non-human mammals, for example, the universal CAR-T cells provided herein can be used for human clinical studies and treatments after their safety and efficacy are first validated in animal models.

In one specific embodiment, we devised the following general CAR-T treatment strategy (described below as dasatinib as an example of a T cell activity inhibitor): (1) The dasatinib and the general CAR-T are combined, and the dasatinib inhibits the activities of host T cells and NK cells, so that the dasatinib is prevented from killing the general CAR-T; (2) Because dasatinib is also able to inhibit universal CAR-T activity, engineering universal CAR-T to be resistant to dasatinib; (3) Modification of universal CAR-T uses a CRSIPR-Cas 9-related cytosine base editor (CBE 3) to point mutate its Lck gene, resulting in Lck protein T316I mutation; (4) The mutated UCAR-T T316I cells were able to demonstrate tolerance to dasatinib drugs. Therefore, under the treatment of dasatinib, T cells and NK cells of a host are inhibited, foreign cells are not cleared, and UCAR-T can perform normal tumor killing function and normal amplification; (5) In addition to editing the Lck gene, the TRAC gene for CAR-T cells is still required in this strategy to avoid GvHD.

The strategy mechanism of the universal CAR-T in the case of dasatinib effectively inhibiting host T cell activity is shown in figure 1. This strategy was designed based on the ability of dasatinib to effect clearance of host T cells, the primary concern being clearance of UCAR-T by host T cells. Therefore, in order to avoid the possibility of eliminating UCAR-T by host NK cells, we did not knock out HLA-I class molecules, and directly avoid host NK cell activation caused by HLA-I class molecule knockout.

As shown in FIG. 1, the leftmost column shows the reason why UCAR-T cells are cleared in vivo by host T cells and are difficult to expand after normal host reinfusion. The TCR of the host T cell recognizes the mismatch of HLA-I and II molecules on the surface of UCAR-T cell, mainly the mismatch of HLA-I molecules recognized by cytotoxic CD8 positive T cells, and the hostAfter autophosphorylation of the intracellular tyrosine kinase LCK of T cells, the TCR and CD3 intracellular segment ITAMS signals are phosphorylated, thereby activating T cells and clearing UCAR-T. The middle column shows that dasatinib, when used in combination, inhibits host T cell TCR activation by inhibiting LCK autophosphorylation of the host T cell. Whereas for universal CAR-T cells, dasatinib also inhibits phosphorylation and activation of CAR molecules by inhibiting LCK autophosphorylation, resulting in the universal CAR-T not being effectively activated by tumor cells for expansion. The right-most column shows single base mutation of LCK to universal CAR-T, allowing LCK ^m Resisting the binding of dasatinib, resulting in drug tolerance. Then, under combined dasatinib treatment, the edited LCK ^m UCAR-T cells are normally activated by tumor cells and effectively expanded.

The strategy mechanism of the universal CAR-T in the case of dasatinib effectively inhibiting host NK cells is shown in figure 2. This strategy was designed based on the ability of dasatinib to effect clearance of host NK cells, which is mainly considered for UCAR-T clearance. Therefore, in order to avoid the damage of host T cells (mainly CD8 positive cytotoxic T cells) to UCAR-T, the B2M gene is knocked out, so that the UCAR-T does not express HLA-I molecules, and the activation of the host T cells caused by mismatch of the HLA-I molecules is directly avoided.

As shown in FIG. 2, the leftmost column shows the principle of clearance of UCAR-T cells by NK cells in vivo after normal reinfusion into the host. Since HLA-I class molecules are the primary ligands for NK inhibitory receptors, B2M knockdown would render the NK inhibitory receptors unrecognizable as ligands, LCK autophosphorylation activates NK, mediating killing of UCAR-T. The middle column shows that when dasatinib is added, the activities of host NK and UCAR-T are inhibited, the host NK does not clear UCAR-T cells, and the UCAR-T cells also have no tumor killing function. The right-most column shows when UCAR-T develops LCK ^T316I After mutation, UCAR-T showed resistance to dasatinib binding, thus host NK cell activity was inhibited in the presence of dasatinib treatment, whereas LCK after editing ^m UCAR-T cells are normally activated by tumor cells and effectively expanded.

LCK function in T cell signaling

LCK is a member of the Src family of kinases and generally binds to the intracellular region of the CD4, CD8 co-receptor molecule of T cells, mediating phosphorylation of the intracellular segment of the T cell receptor molecule, ITAMS. LCK is therefore the most upstream tyrosine protein kinase for TCR signaling, mediating the transmission of the first signal upon antigen stimulation of T cells.

As shown in FIG. 3, when T cells are stimulated with antigen-MHC molecule complexes of antigen presenting cells, LCK proteins bound to the intracellular segment of the co-receptor autophosphorylate to become activated phosphorylated LCK, which in turn phosphorylates ITAMS of the intracellular segments of TCR and CD3 molecules. At the same time, phosphorylated SH2 domains recruit ZAP70, LCK further phosphorylates ZAP70, and MEK/ERK pathways are under activation, which participate in T cell activation. LCK is thus the most upstream and critical loop in the activation pathway of T cells.

Inhibition of T cell function by dasatinib

To date, inhibition of T cell function by dasatinib has been an accepted event. Two documents in 2008 respectively describe how dasatinib inhibits T cell function in detail [ ^1,2 The process is carried out. Stephen Blake et al reported that dasatinib as an inhibitor of Src/ABL kinase is effective in inhibiting many functions of normal human T lymphocytes in vitro, including that dasatinib can effectively block TCR signaling transduction by binding LCK, inhibit T cell activation, cytokine secretion and proliferation in vitro, but does not affect T cell viability [ ¹ The process is carried out. Similar experimental results are reported by Andrew e.schade et al, which considers dasatinib to inhibit TCR-mediated signal transduction, cell proliferation, cytokine secretion and cellular responses in vivo by inhibiting LCK phosphorylation. However, this inhibition was reversible, and the function of T cells was restored when dasatinib was removed [ ² 】。

Inhibition of CAR-T cell function by dasatinib

Since dasatinib is able to inhibit LCK phosphorylation in general to inhibit TCR function, then CAR-T signaling would be inhibited by dasatinib? In the year 2019,there are two documents detailing how dasatinib inhibits CAR-T cell function, respectively [ ^3,4 The process is carried out. Evan W.Weber et al reported that dasatinib is a potential, rapid, reversible inhibitor of CAR-T cell function, capable of inhibiting CAR-T cell proliferation, cytokine secretion and tumor killing activity in vivo [ ³ The process is carried out. Katrin Mestermann et al reported that dasatinib was able to bind to LCK, inhibiting phosphorylation of CD3z and phosphorylation of ZAP70, and thus dasatinib was able to inhibit activation of cd28_cd3z or 4-1bb_cd3z in the CAR molecular structure to inhibit CAR molecular function. And dasatinib induces CD8 ⁺ And CD4 ⁺ The functional resting state of CAR-T can last for several days without affecting T cell viability. The data indicate that dasatinib is able to inhibit CAR-T cell secretion cytokines and proliferation in vivo and in vitro, and that this inhibition is reversible [ ⁴ 】。

Inhibition of NK cell function by dasatinib

Dasatinib has the function of inhibiting T cells and CAR-T cells, and can inhibit degranulation and cytokine release of primary NK cells [ ⁵ 】。

Relationship of LCK mutations to dasatinib tolerance

It has been shown that LCK-T316M mutations can exhibit resistance to dasatinib [ ⁶ The principle is that the T316 site is a key gatekeeper amino acid of LCK kinase, and the mutation of T316M leads to great change of LCK structure. As shown in fig. 4, dasatinib was able to bind to the ATP binding region of LCK when position 316 was T, inhibiting autophosphorylation of LCK, whereas dasatinib was blocked off the door from binding to LCK-T316M when position 316 was mutated to M.

As small molecule inhibitors targeting ABL kinase, dasatinib has many analogues such as Imatinib and nilotinib, etc. Among them, the most studied is the relationship between mutation of ABL and Imatinib tolerance. The most common ABL mutations that can cause Imatinib tolerance are T315I, T315A, etc. [ ^7,8,9 The process is carried out. We found that the activation domains between ABL kinase and LCK kinase are very similar, as shown in FIG. 5, the amino acid sequences before and after ABL-T315 and LCK-T316Highly conserved, we speculate that LCK-T316I and LCK-T316A may also be tolerant to dasatinib. Therefore, subsequent LCK mutations we can design from three directions, T316M/A/I.

Design for inducing LCK mutations on T cells

CRISPR-Cas9 is used as one of the common gene editing tools at present, and can realize specific and efficient cutting on genome under the mediation of single-stranded targeted RNA. Thus, CRISPR-Cas 9-related gene editing is well suited for the induction of LCK-T316 point mutations. The following two sets of CRISPR related technical schemes are designed, and mutation of LCK-T316 locus on T cells can be realized.

Strategy for inducing LCK-T316M/I/A mutation based on CRISPR-Cas9 mediated homologous recombination repair

CRISPR-Cas9 mediated repair of homologous recombination is a very effective technical means to achieve base/amino acid point mutations. The principle is that after the Cas9 protein and the sgRNA form RNP, the RNP can be targeted to a specific position of a genome by the sgRNA, and the cutting is carried out at a position 3-4nt in front of a PAM sequence to induce double-stranded DNA to break. At this time, the introduction of homologous recombination carrying LCK-T316 site mutation can allow the cell to repair with the template sequence, thereby obtaining endogenous LCK-T316 mutated cell. The template for this homologous recombination may be double-stranded DNA or single-stranded DNA. In the point mutation induction system, the efficiency of single-stranded DNA template mediated homologous recombination is highest.

Thus, we originally designed 16 sgrnas for Cas9 systems for a length of 100bp around the LCK-T316 site. As shown in FIG. 6, only three sgRNAs of sg8, 9 and 12 were cut at 7-12nt from the mutation site near T316, and the other sgRNAs were cut at too far from the mutation site, so that the efficiency of homologous recombination was significantly reduced, and therefore we selected three sgRNAs of sg8, 9 and 12 to evaluate the cutting efficiency and off-target condition. The specific sequences of the three sgrnas are set forth in SEQ ID NOs: 16. 17 and 18, the corresponding ssDNA-induced T316I mutation sequences are set forth in SEQ ID NOs:19-21, the corresponding ssDNA-induced T316M mutation sequence is set forth in SEQ ID NO:22-24, the corresponding ssDNA inducing T316A mutation sequence is shown in SEQ ID NOs:25-27.

The off-target situation is shown in FIG. 7, where there are 3, 2 and 3 MM2 for sg8, 9 and 12 respectively. The number of MM2 indicates how many sites on the whole genome there are and only 2 mismatches with the sgRNA. In general, special care needs to be taken when MM0, MM1 and MM2 occur to off-target this sgRNA. Thus, we carefully examined the off-target sites of sg8, 9 and 12, and found that sg8 was most likely to off-target to the exons of the HCK and PDGFRB genes, sg9 was most likely to off-target to the exons of the HCK and FYN genes, and sg12 was most likely to off-target to the exons of the DDR2 genes. In the case of these off-targets, cas9 proteins are able to cleave off the off-target region, thus knocking out the relevant gene, resulting in a significant risk of off-target. Thus, although this scheme can induce mutation of T316 to M/I/A and the like in various amino acid directions, we have not finally selected the scheme.

Wherein the LCK-T316I protein sequence is shown in SEQ ID NO 9, the LCK-T316A protein sequence is shown in SEQ ID NO 10, and the LCK-T316M protein sequence is shown in SEQ ID NO 11.

Another method for inducing LCK-T316M/I/A mutation by CRISPR-Cas9 mediated homologous recombination is repair by two sgRNA cleavage plus single stranded ssDNA templates. Induction of T316I/M/A mutations can also be achieved by either sgRNA1 (SEQ ID NO: 14) in combination with sgRNA11 (SEQ ID NO: 15) or sgRNA1 in combination with sgRNA12 (SEQ ID NO: 18). Wherein, the T316I/M/A mutant ssDNA template sequences required by the sgRNA1 to match the sgRNA11 are respectively shown in SEQ ID NOs:28-30, the T316I/M/A mutant ssDNA template sequences required for the sgRNA to match the sgRNA12 are respectively shown in SEQ ID NOs:31-33.

CRISPR-Cas 9-based cytosine base editor CBE3 induced LCK-T316I mutation strategy

Another tool capable of inducing LCK-T316 mutations is a cytosine base editor based on CRISPR-Cas9 technology. The principle of CBE3 (cytosine base editor 3) is that Cas9n protein and cytosine deamination-inducing protein APOBEC-3A are fused and expressed (A3A-CBE 3), and when Cas9n cuts a single chain at a target site, cytosine deamination is induced to generate mutation. Cas9n is prepared by D10A mutation of RuvC1 domain of Cas9 protein to become Cas 9-nicase protein (Cas 9 n), and only the enzyme activity of HNH domain is reserved. Cas9n does not cause DNA double strand breaks, but is only able to cleave DNA single strands that bind complementarily to sgrnas on the genome, thereby inducing base mismatch repair (BER). At this time, APOBEC-3A can induce deamination of cytosine on another DNA single strand to form uracil, which finally promotes mutation of cytosine to thymine (C- > T mutation) in the presence of UGI protein.

The inventor clones A3A-CBE3 gene of an escherichia coli expression system, and expresses functional A3A-CBE3 protein through escherichia coli purification, and the protein sequence is shown in SEQ ID NO:34.

as shown in FIG. 8, we searched for two sgRNAs-sg 12 and sg16, which were consistent with cytosine base editing, from the sgRNAs near the LCK-T316 site. When T316 (ACT) is base edited, its second codon cytosine (C) is directionally mutated to thymine (T) and can induce the formation of I316 (ATT).

This system has the advantage over Cas 9-mediated homologous recombination schemes: (1) Since Cas9n protein is used in CBE3 system, double-stranded DNA cleavage of genome is not performed, so risk due to sgRNA off-target in this set of system is very low; (2) The gene editing can be realized by directly electrotransferring RNP formed by CBE3 protein and sgRNA without template ssDNA; (3) Most importantly, although CBE3 base editing cannot be precisely targeted at the fixed point of a single base within the coverage of sgRNA at present, any mutation of cytosine except the T316 site under the coverage of the sgRNA does not cause amino acid mutation near the LCK-T316 site, namely, the CBE3 strategy only causes single amino acid mutation of T316I.

There are two problems with this strategy that remain to be validated:

(1) The designed sg12 and sg16 sgRNAs can efficiently induce LCK-T316I mutation;

(2) LCK-T316I mutations can cause their tolerance to dasatinib.

Based on this, we optimized the length of sg12 and sg16, as shown in table 1, and designed a total of 7 sgrnas for subsequent mutation induction and functional verification.

TABLE 1LCK-T316I mutant sg12 and sg16 sequence listing

The inventors also designed LCK-sg16-19nt (TCACTGAATACATGGAGAA, SEQ ID NO: 7) as LCK-T316I mutated sgRNA, but did not verify experimentally, and theorized that 19nt length sgRNA and 18nt, 20nt and 21nt had similar or identical functions.

In addition, the inventors have designed a useful and applicable sa-sgrnas for saCas 9-induced base editing, two in total, LCK-saCas9-sgRNA1 and sgRNA2, respectively, with sequences as set forth in SEQ ID NOs:12 and 13.

Functional verification of LCK-T316I mutation induction by sgRNA

sgRNA16 can efficiently induce LCK-T316I mutation

We isolated CD3 positive T cells from cryopreserved PBMC, and activated CD3/CD28 DynaBeads for 48hr after which RNP complexes of CBE3 protein and sgRNA were used for electrotransformation, and the T cell genome was extracted 96hr after editing, and LCK gene editing efficiency was examined by PCR and Sanger sequencing.

As described in detail in the examples below, T cells that were electroporated with the CBE3 protein group only did not have any base mutation in the Lck gene region of the genome, whereas the RNPs formed by the different lengths of LCK-sgRNA16 and CBE3 proteins were able to cause T316I mutation with a mutation efficiency of approximately 50%. Meanwhile, the four cytosine residues in the front of the T316 locus are mutated in C- > T, but all the cytosine residues do not cause amino acid changes. Thus, in T cells, all three different lengths of LCK-sgRNA16 were able to induce LCK-T316I mutations with high efficiency. To reduce the impact of sgRNA off-target on editing, we biased to follow-up experiments with 21nt of sgRNA 16.

The sgRNA12 was unable to induce LCK-T316I mutation

As in the gene editing experiments described above, we also examined the efficiency of sgRNA12 induction of T cell LCK-T316 mutation. Compared with the control CBE3, LCK-sgRNA12 with different lengths has no edit on cytosine at the T316 locus, can induce about 50% C- > T mutation on four cytosine before the T316 locus, but can not cause any change of amino acid. Therefore, LCK-sgRNA12 was not effective in inducing LCK-T316I mutation, but could be used as a negative control for sgRNA16, so we selected 21nt LCK-sgRNA16 as a control for subsequent experiments.

Relationship verification of LCK-T316I mutation and dasatinib inhibition T/CAR-T signal activationPre-experiment verification of dasatide Nifunction, determining dasatinib to be highly effective in inhibiting T cell TCR activation and NK cell CD107a transport

To verify the inhibition of T cell TCR signaling activation by dasatinib, we treated T cells with dasatinib or DMSO for 5 hours in advance, then activated T cells with CD3/CD28 DynaBeads for 24 hours, then examined the expression of the activating molecules CD25, CD69 and 4-1BB by flow. The results showed that the DMSO control group significantly upregulated the activating molecule CD25 24 hours after bead activation, while CD69 positive or 4-1BB positive cells were significantly clustered. And 100nM/1000nM dasatinib treated, again with CD3/CD28 activation also completely without CD69/4-1BB positive activated cell population, suggesting that 100nM dasatinib has been able to effectively inhibit TCR signal activation.

Next, we performed culture expansion of NK cells isolated from human PBMC in vitro in order to verify inhibition of NK cell activation by dasatinib. Treatment with different concentrations of dasatinib for 24hr, one group was inactivated and the other group was stimulated with NK-activating target cells K562 for 5 hours prior to detection of CD107a, followed by uniform detection of NK cell CD107a transport. The results show that the basal CD107a trafficking was also reduced to 2% baseline after treatment with dasatinib for the non-activated group of NK cells. While following K562 target cell activation, untreated group CD107a transport was 64.5% and treated group was reduced to 2% baseline, suggesting that 100nM dasatinib was sufficient to inhibit NK cell CD107a transport, inhibiting NK cell activation.

LCK-T316I can make T cell resistant to inhibition of TCR activation by dasatinib

To verify the tolerance of LCK-T316I mutations to dasatinib function, we performed the following experiments on LCK-edited T cells. Three different sets of treatments were set up for each set of edit cells and MockT cells: the first group was not added with dasatinib and activated with CD3/CD28 magnetic beads; the second group was not added with dasatinib, but TCR activation was performed with CD3/CD28 magnetic beads for 24 hours; the third group was pre-treated with 100nM dasatinib for 5 hours, after which CD3/CD28 beads were added for TCR activation for 24 hours, and the effect of gene editing on dasatinib inhibition of TCR activation was observed.

The results show that the CD25/CD69 positive cell proportion in the control group, plus 100nM dasatinib, was significantly lower than in the active group, indicating that dasatinib inhibited CD3/CD28 activation of TCR. Meanwhile, in the sg16 edited group, a high proportion of CD25/CD69 positive cells still exist in the dasatinib group, and the same result can be obtained when 4-1BB is used as an activation marker. Thus, a fraction of the cells after sg16 editing could still be activated to CD69/4-1BB positive after dasatinib treatment, demonstrating that they were able to tolerate inhibition of T cell activation by dasatinib.

We further counted the proportion of CD25, CD69 and 4-1BB single positive cells in the control, sg12 and sg16 edited groups, and found that the expression proportion of the three activation markers was significantly increased in the sg16 edited group under treatment with dasatinib and CD3/CD28 magnetic beads compared to the control and sg12 edited groups. It was further suggested that the sg 16-edited group of T cells (-50% LCK-T316I) showed tolerance to dasatinib, capable of activation by CD3/CD 28.

LCK-T316I renders anti-CD19-CAR-T cells resistant to inhibition of CD107a transport by dasatinib

To verify that LCK-T316I mutations not only enable T cells to tolerate inhibition of TCR activation by dasatinib, but also CAR-T cells to tolerate inhibition of CD107a transport by dasatinib during their CAR activation, we designed the following experiment. Firstly, three groups of anti-CD19 CAR-T are subjected to different editing treatment of CBE3 protein, CBE3+sg12 and CBE3+sg16, after 72hr stabilization after editing, each group of CAR-T cells are subjected to pretreatment of 100nM dasatinib for 12 hours, different target cells are added for stimulation, CAR signals are activated, and CD107a release in CAR-T killing target cells is mediated. 5 hours prior to detection, monensin and CD107a antibodies were added, and after 5 hours, staining was performed for CD8 and CAR positivity for detection of enrichment of CD107a transport of CD8 positive CAR positive cells.

The sequences of the anti-CD19-CAR molecules used herein are identical to those used in the examples that follow, the specific sequences being set forth in SEQ ID NOs:35-56. The overall structure of this CAR molecule is a second generation CAR molecule consisting of an anti-CD19 scFv (SEQ ID NOs: 35-38), a junction region (SEQ ID NOs: 39-40), a CD8ahinge region (SEQ ID NOs: 41-42), a CD8a transmembrane domain (SEQ ID NOs: 43-44), a CD28 intracellular domain (SEQ ID NOs: 45-46), a CD3z intracellular signaling domain (SEQ ID NOs: 47-48), a T2A sequence (SEQ ID NOs: 49-50), a CSF2RA signal (SEQ ID NOs: 51-52), and a tEGFR signal (SEQ ID NOs: 53-54). The total sequence of the final anti-CD 19-CD28z-CAR is set forth in SEQ ID NOs:55-56.

The results show that in the DMSO control group, the negative target cell K562 can not activate the CAR-T to carry out effective CD107a transportation no matter whether the anti-CD19-CAR-T cells are edited or not, while the positive target cell Nalm6 can fully activate the CAR-T to release the CD107a, and the positive rate is more than 70%. Whereas negative control group K562 showed little enrichment of CD107a transport at 100nM dasatinib, the positive target cell Nalm6 stimulated only the sg16-LCK-T316I edited group showed 50% positive CD107a transport, suggesting that LCK-T316I mutation did indeed allow anti-CD19-CAR-T cells to tolerate inhibition of CD107a transport by dasatinib with a significant difference.

LCK-T316I renders anti-BCMA-CAR-T cells resistant to inhibition of CD107a transport by dasatinib

To verify that LCK-T316I mutations can render other CAR-T cells resistant to inhibition of CD107a transport by dasatinib in addition to anti-CD19 CAR-T, we performed three sets of edits of LCK-CBE 3, cbe3+lck-sg12 and cbe3+lck-sg16 for anti-BCMA CAR-T, the experimental procedure was fully consistent with that described above. Three groups of target cells used to stimulate anti-BCMA CAR-T were K562 negative target cells, U266-B1 positive target cells, and RPMI-8226 positive target cells, respectively. The results show that in the DMSO-treated control groups, three edited anti-BCMA CAR-T had higher levels of CD107a transport under stimulation of positive target cells, while in 100nM dasatinib treatment, only the LCK-T316I mutant group (cbe3+sg 16 group) showed tolerance to dasatinib, with 39.6% and 44.7% positive CD107a release, respectively, under activation of both positive target cells. At the same time, there was a significant difference between this degree of CD107a release and other editorial control groups, suggesting that LCK-T316I mutations could render anti-BCMA-CAR-T cells resistant to inhibition of CD107a transport by dasatinib.

LCK-T316I makes anti-CD19/BCMA double-target UCAR-T cell resistant to inhibition of CD107a transport by dasatinib Manufacturing process

Furthermore, in substantial agreement with the above experiments, we further validated the inhibition of CD107a transport by LCK-T316I mutation on this kind of CAR-T cells tolerating dasatinib in anti-CD19 and BCMA dual-target UCAR-T cells (knockdown of TRAC). The results show that under normal conditions, negative target cell K562 was substantially inactive against double UCAR-T, and that both positive target cells U266 and Raji were able to activate double UCAR-T. At 100nM dasatinib treatment, only the sg 16-edited group was able to demonstrate resistance to dasatinib inhibiting the transport of CD107a under activation of target cells, and the foregoing results were fully met.

Relationship verification of LCK-T316I mutation and dasatinib for inhibiting CAR-T killing function

LCK-T316I enables anti-BCMA Inhibition of CAR-T killing function by CAR-T cell-tolerant dasatinib

It was mentioned above that mutation of LCK-T316I can make T cells resistant to inhibition of TCR activation by dasatinib, while also making CAR-T cells resistant to inhibition of CAR activation by dasatinib and inhibition of CD107a transport. We have further explored whether this tolerance is reflected in T cell function, and have therefore devised the following experiments to explore the relationship between LCK-T316I mutation and dasatinib's inhibition of CAR-T killing function.

Three different groups of CBE3 protein, CBE3+sg12 and CBE3+sg16 were edited by our anti-BCMA CAR-T, after 72hr stabilization of the edits we pre-treated each group of CAR-T cells with 100nM dasatinib for 12 hours, followed by and different luciferase positive target cells (luciferases+) to target ratio 1:1 for 24 hours, and after the incubation, the killing of the target cells by CAR-T cells was assessed by detecting luciferase activity, the lower the luciferase number, the less viable target cells, the stronger the CAR-T killing. The result shows that in the DMSO control group, the anti-BCMA CAR-T can cause the reduction of the luciferase value of the RPMI-8226 positive target cells, but the luciferase value of the negative target cells Nalm6 is not influenced, and the killing activity of the anti-BCMA CAR-T is proved to be normal. Under the stimulation of positive target cells RPMI-8226 under the treatment of 100nM dasatinib, the luciferase values in the CBE3 and CBE3+sg12-LCK unedited groups are obviously increased compared with the DMSO group, and the killing function of the anti-BCMA CAR-T is obviously inhibited under the treatment of dasatinib. And the luciferase value in the CBE+sg16-LCK editing group is obviously lower than that in the CBE3 and CBE3+sg12-LCK unedited group, so that the anti-BCMA CAR-T after LCK-T316I mutation is proved to have tolerance on the CART killing activity inhibition function of dasatinib.

In addition, we repeatedly edited another lot of anti-BCMA CAR-T cells, and also obtained results consistent with the above experiments. In this repeat we used two other negative target cells, K562-luciferase and Raji-luciferase. The Luciferase results show that in the DMSO control group, all edited anti-BCMA CAR-T cells can not kill both K562 and Raji negative target cells, but target RPMI-8226 positive target cells, and the Luciferase value is basically reduced to a baseline, so that the anti-BCMA CAR-T has normal specific killing function. Subsequently, higher luciferase values were still detectable in the CBE3 and cbe3+sg12 groups of positive target cells with 100nM dasatinib treatment, suggesting that the killing function of CAR-T was still inhibited. While the luciferase values of the cbe3+sg16-LCK-T316I edited group were significantly reduced, again demonstrating the tolerance of LCK-T316I mutations to the CAR-T killing inhibitory function of dasatinib.

The CAR molecule sequences of the anti-BCMA-CAR-T cells used herein and in the examples that follow are identical, the specific sequences are set forth in SEQ ID NOs:77-78.

Relationship verification of LCK-T316I mutation and dasatinib inhibition of T/CAR-T cell proliferation

LCK-T316I enables anti-BCMA Inhibition of cell proliferation by CAR-T cells tolerating dasatinib

Since LCK-T316I is tolerant to the TCR/CAR activation inhibition of dasatinib and the killing of CAR-T cells, we want to further explore whether the inhibition of T/CAR-T proliferation in vitro by dasatinib is resisted in LCK-T316I mutated CAR-T cells. We have therefore devised the following experiments to verify CAR-T proliferation in vitro. Firstly, three kinds of editing including CBE3, CBE3+sg12 and CBE3+sg16 are carried out on anti-BCMA CAR-T cells, and after the editing is stabilized for 72 hours, the effective target ratio is 10:1 target cell U266 was added for stimulation every 3 days while total number of cells and CAR positive rate were detected every three days. The experimental procedure was divided into two groups with DMSO and 100nM dasatinib. As shown in fig. 9, the results showed that although CAR-T cells in DMSO group did not expand well, differences in CAR positive rates of cbe3+sg16 and the other two groups were shown in Day12 in dasatinib treated group, while the total number of cells of the whole group was also the greatest. Preliminary suggestion that LCK-T316I was able to make anti-BCMA CAR-T cells tolerant to inhibition of cell proliferation by dasatinib.

Preliminary brief summary of data

The method for carrying out site-directed mutagenesis on LCK-T316I is found, namely, a cytosine base editor CBE3 is matched with LCK-sgRNA16, and efficient LCK-T316I mutation of T cells is realized through electrotransformation;

LCK-T316I mutations are able to resist many functions of dasatinib on T cells:

dasatinib can inhibit activation of CD3/CD28 to TCR signal with high efficiency;

dasatinib can inhibit activation of target cells to CAR-T cells with high efficiency;

LCK ^T316I tolerating T cells to inhibition of TCR activation by dasatinib;

LCK ^T316I tolerating the CAR-T cells to inhibition of CAR activation by dasatinib;

LCK ^T316I tolerating anti-CD19-CAR-T cells to inhibition of CD107a transport by dasatinib;

LCK ^T316I tolerating anti-BCMA-CAR-T cells to inhibition of CD107a transport by dasatinib;

LCK ^T316I tolerating anti-CD19/BCMA dual-target CAR-T cells to inhibit CD107a transport by dasatinib;

LCK ^T316I tolerating the CAR-T cells to inhibition of CAR-T killing function by dasatinib;

LCK ^T316I the CAR-T cells were made resistant to inhibition of cell proliferation by dasatinib.

In addition, we further studied to find that:

dasatinib at a concentration of 25nM is effective to inhibit T cell activation in vitro;

in vitro dasatinib has a continuous effect on inhibiting T cell activation function and has LCK ^T316I The mutated CD5-UCART can tolerate such inhibition;

dasatinib can effectively inhibit activation of NK cells;

dasatinib at a concentration of 30nM is effective in inhibiting NK cell activation in vitro;

the inhibition of NK cell activation function by in vitro dasatinib has a continuous effect;

LCK ^T316I The combined strategy of mutation and dasatinib enables the CD5-UCART to have the advantages of amplification and persistence in vitro mixed lymphocyte reaction with 5 to 1 effective target ratio;

LCK ^T316I the mutation and dasatinib combined strategy enables the CD5-UCART to have the advantages of amplification and persistence in 50-100 to 1 effective target ratio in vitro mixed lymphocyte reaction;

panatinib (PN) at a concentration of 200nM is effective in inhibiting T cell activation in vitro, while LCK ^T316I Mutations can tolerate such inhibition;

panatinib at a concentration of 200nM is effective to inhibit NK cell activation in vitro;

the inhibition of T cell activation function by panatinib in vitro has a continuous effect;

the inhibition of NK cell activation function by panatinib in vitro has a continuous effect;

verifying the possibility of panatinib as a combination drug and switch for the universal CAR-T treatment of the present patent;

it was demonstrated that the universal CAR-T cells with B2M knockdown were resistant to NK cell killing in the strategy of the invention.

Finally, after LCK-T316I editing is carried out on the general CAR-T and dasatinib/panatinib treatment is matched, the activation and killing functions of the host T and NK are inhibited, so that LCK ^T316I The UCAR-T of the strain is not cleared by the immune system of a host, has normal tumor killing function, can be effectively amplified in vivo and plays a role.

The invention is further illustrated by the following specific examples.

Example 1: methods and materials

Method

1.1 Sorting and activation of CD3+ T cells

Resuscitating cryopreserved healthy donor PBMC 1.0X10 total ⁸ Cells were resuspended in 8mL of pre-warmed ringing buffer after flash thawing per tube, and small amounts of cell suspension were taken for cell counting. PBMC suspension was centrifuged at 400g, at an ascending rate of 8, at a descending rate of 8 (hereinafter +.8) and for 10 minutes. After centrifugation, the supernatant was discarded and 20ul/10 was added ⁷ After being mixed uniformly, the CD3 microbeads are put into a refrigerator with the temperature of 4 ℃ for incubation for 20 minutes, and the wall of the tube is flicked for a plurality of times every 10 minutes to avoid cell precipitation. After the incubation, the cells were resuspended by adding a binding buffer, rinsing 1 time, centrifuging (400 g 10min +.8 ≡8), and then 500. Mu.l of binding buffer. Meanwhile, the LS sorting column is placed on a Meitian gentle magnetic sorting frame, after the LS sorting column is rinsed and washed for 1 time by 2ml Rinsing buffer, 500 mu l of cell suspension is added, and after the cell suspension is completely dripped, the cell suspension is repeatedly added on the LS column for 2 times, and 2ml Rinsing buffer times. The target cells were washed from the LS column with 5mL Rinsing buffer and collected, and after appropriate dilution, the target cells were counted to about 1X 10 ⁵ Individual cells were flow cytometry to determine the purity of the sorted T cells. Subsequently 300g of the cell suspension was centrifuged for 10 minutes and the cell density was adjusted to 1X 10 with fresh T cell medium ⁶ At 10ul/10 ⁶ Concentration of individual cells activated by addition of anti-CD 3/-CD28 antibody magnetic beads, seeded into 12-well plates at 4mL per wellPlacing at 37deg.C, CO ₂ Culturing in an incubator.

1.2 T cell CAR positive lentiviral transduction

The CD3 positive T cells were activated with anti-CD 3/CD28 magnetic beads for 24-48 hours and virus transduction was performed. Cell suspensions were subjected to viability assay and cell counting. Cells were collected by centrifugation at 300g for 10 minutes and the cell density was adjusted to 1X 10 ⁶ 1mL volume per well in 24 well plates, lentivirus was added, MOI adjusted to 3, 800ng/uL PolyBrene and 1ug/uL DEAE were added to assist in the transfer. After mixing, the culture was continued in an incubator at 37 ℃. After 24 hours, the virus was removed by centrifugation at 300g for 10 minutes, and T cells were cultured.

1.3 T cell gene knockout

Taking TRAC and CD5 knockouts as examples: after T cells were activated with anti-CD 3/anti-CD 28 antibody magnetic beads for 24 hours, the magnetic beads were removed, and the cells were counted and the cell viability was confirmed, taking several 2X 10 portions ⁶ The cell suspension was centrifuged at 100g for 10 min in a centrifuge tube. After centrifugation, the medium was completely removed and resuspended with Lonza electroporation buffer. At the same time, 35pmol of Cas9 protein+75 pmol of TRAC-sgRNA was prepared for each serving; 35pmol of Cas9 protein+60 pmol of CD5-sgRNA RNP complex were incubated at room temperature for 15 minutes, respectively. Then mixing the cell suspension with RNP complex, adding into Lonza 16-hole electric rotating cup, and placing into 4D-Nucleofector ^TM The X units were electroporated as EH-115 program. 80 uL/well pre-incubation was added to the electrorotor for 15 minutes based on 37 degrees, after which the culture was continued with 500uL T cell complete medium in 48 well plates.

1.4 LCK-T316I Gene editing (Km editing introduction)

T cells were taken, 2X 10 in each group ⁶ The cell suspension was centrifuged at 100g for 10 min in a centrifuge tube. After centrifugation, the medium was completely removed and resuspended with Lonza electroporation buffer. At the same time, RNP complexes for the above groups (per part) were prepared: (1) 60pmol CBE3 protein; (2) 60pmol CBE3 protein+100 pmol K-sgRNA12; (3) 60pmol CBE3 protein+100 pmol K-sgRNA16 RNP complex were incubated at room temperature for 15 min. Subsequently complexing the cell suspension with RNPMixing the above materials, adding into Lonza 16 hole electric rotating cup, and adding into 4D-Nucleofector ^TM The X units were electroporated as EH-115 program. 80uL of pre-incubation was added to the electrorotor for 15 minutes based on 37 degrees, after which the culture was continued in 48 well plates with 500uL of T cell complete medium.

1.5 Gene knockout efficiency detection

Taking TRAC and CD5 knockouts as examples: TRAC and CD5 gene knockout efficiency was examined by flow cytometry 72 hours after electrotransfection of the T cells with TRAC and CD5 genes. The method comprises the following specific steps: about 2X 10 ⁵ The individual cells were taken as cells in 1.5mL centrifuge tubes, washed 2 times with PBS+2% fetal bovine serum buffer, the supernatant was completely discarded, the cells were resuspended with 100. Mu.L buffer, 1. Mu.L each of PE-anti-human-TRAC and APC-anti-human-CD 5 antibodies was added, mixed well, incubated at 4℃for 30 minutes in the absence of light, washed once with buffer, and then checked on a machine.

1.6 CAR positive rate detection

CAR positive rate was detected by flow cytometry 5 days after T cell infection with lentivirus. The method comprises the following specific steps: about 1X 10 in two portions ⁵ Individual cells were taken as cells in 1.5mL centrifuge tubes and washed 2 times with PBS +2% fetal bovine serum buffer and the supernatant was completely discarded. One part was resuspended in 100. Mu.l buffer followed by 5. Mu.l FITC-labeled-human-target antigen protein and the other part was resuspended in 100. Mu.l buffer followed by 1. Mu.l APC-anti-human-EGFR (transduction-marker) antibody (if EGFR expression) and incubated at 4℃for 30 min in the absence of light after homogenization, washed once with buffer and checked on the machine.

1.7 detection of the edit efficiency of LCK-T316I Gene editing

The T316I mutation rate was detected 72 hours after Km editing of T cells or CAR-T cells. Taking about 1×10 groups ⁵ Each cell was subjected to extraction of genomic DNA, and a DNA fragment covering the Km region and containing about 200bp sequences upstream and downstream was amplified by PCR using the genomic DNA as a template. The nucleotide sequence was obtained by Sanger sequencing. Uploading Sanger sequenced ab1 format file in edition of EditR webpage, inputting 20bp sgRNA sequence, clicking data QC and pre edition to watch analysis result, and communicating Downloaded over Download Report.

The main reagent materials used are listed in table 2.

TABLE 2 Main reagent materials

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Example 2: ^T316I efficient induction of human LCK mutations in T cells using the single base editor A3A-CBE3(abbreviated as Km)

The inventors utilized single base cytosine editor A3A-CBE3 based on CRISPR-Cas9 technology ¹⁰ ]T316I mutation is induced on human LCK protein, namely, the 316 th amino acid of human LCK protein is mutated from threonine (T) to isoleucine (I), which is abbreviated as Km.

The A3A-CBE3 protein is a nickase fusion protein of APOBEC3A and Cas9-D10A mutation, can be targeted to a specific position of a genome by sgRNA of a specific sequence, and mediates mutation of cytosine to thymine in the targeting range of the sgRNA, thereby realizing base editing and protein mutation. Cas9-D10A is prepared by carrying out D10A mutation on the RuvC1 domain of Cas9 protein to form Cas9-nickase protein (Cas 9 n), and only retains the enzyme activity of HNH domain, so that Cas9n can not cause DNA double strand break, and can only cleave DNA single strand complementarily combined with sgRNA on genome, thereby inducing base mismatch repair (BER). And at this time, the APOBEC3 can induce cytosine on another DNA single strand to deaminate to form uracil, and finally promote mutation of cytosine to thymine (C- > T mutation) in the presence of UGI protein.

The inventors have aimed at inducing LCK ^T316I Mutant sgrnas were designed.

As shown in FIG. 10A, the inventors designed sgRNAs meeting the cytosine base editing characteristics of the T316 site near the LCK-T316 site by analyzing the gene sequence encoding LCK protein, and finally two sgRNAs meeting the single base editing requirement, namely LCK-sgRNA12 and LCK-sgRNA16. When T316 (ACT) is base edited, its second codon cytosine (C) is directionally mutated to thymine (T) and can induce the formation of I316 (ATT). Meanwhile, the inventors modified the length of the sgrnas in order to increase the efficiency of mutation induction thereof, designed the sgrnas with different lengths of 18nt to 21nt, as shown in fig. 10B.

The inventors validated sgRNA against LCK ^T316I Function and efficiency of mutation induction LCK-sgRNA16 is capable of efficiently inducing LCK-T316I mutation, while LCK-sgRNA12 is incapable of inducing LCK-T316I mutation.

The inventor separates CD3 positive T cells from frozen human PBMC, and the sorting positive rate is more than 95%. The selected CD3 positive T cells were activated 48 hours after stimulation with DynaBeads magnetic beads against CD3/CD28 for mutation induction. After in vitro co-incubation of the A3A-CBE3 protein and LCK-sgRNA16 of different lengths to form RNP complex, the RNP complex was purified by LONZANNucleofector-4D electrotransport and P3 Primary Cell 4D-Nucleofector ^TM The X Kit performs electrotransformation of activated T cells, and the electrotransformation program is EH-115. Culturing was continued for 96 hours after electrotransformation, the T cell genome was extracted, and the efficiency of lck gene editing was examined by PCR and Sanger sequencing.

As shown in FIG. 11A, T cells which had not been electroporated with the A3A-CBE3 protein group without the addition of sgRNA had no base mutation in the LCK gene region of the genome (single peak pattern for each sequencing site), whereas RNPs formed by LCK-sgRNA16 and A3A-CBE3 proteins of different lengths were each capable of causing mutation of T316I (red-colored sequencing peak representing thymine T appeared on the blue sequencing peak of cytosine C at T316 site, forming a mantle peak) with a mutation efficiency of about 50% according to the peak analysis of the mantle peak. Meanwhile, the first four cytosines of the T316 locus are mutated in C- > T, but because the four cytosines are all positioned at the third degenerate codon of the amino acid, the main mutations except the cytosine mutated in the T316 locus do not cause the change of the amino acid.

As shown in FIG. 11B, the inventors pass through EditR [ ¹¹ ]Analysis of sequencing results, statistics of mutation frequencies of cytosine at LCK major mutation sites under the induction of sgRNA with lengths of 18nt, 20nt and 21ntThe results show that the cytosine mutation induction efficiency of the sgrnas with different lengths on the T316 site is basically consistent and is about 50%. Thus, in activated T cells, all three different lengths of LCK-sgRNA16 were able to efficiently induce LCK-T316I mutations. To reduce the impact of sgRNA off-target on editing, we performed subsequent experiments with 21nt of sgRNA 16.

The inventors verified that LCK-sgRNA12 was unable to induce LCK-T316I mutation.

As in the gene editing experiments described above, the inventors also examined the efficiency of LCK-sgRNA12 induction of T cell LCK-T316 mutation. As shown in FIG. 12, compared with the sequencing result of the control group which is electrically transfected with the A3A-CBE3 protein, LCK-sgRNA12 with different lengths has no substantial editing on cytosine at the T316 site, and can induce about 50% C- > T mutation on four cytosine before the T316 site, but cannot cause any change of amino acid. Therefore, LCK-sgRNA12 cannot effectively induce LCK-T316I mutation, but can be used as a negative control of sgRNA16, so that 21nt of LCK-sgRNA12 is selected as a control of a subsequent experiment, and the length of the LCK-sgRNA is ensured to be consistent with that of 16-21 nt.

Example 3: verification that dasatinib is effective in inhibiting TCR signaling to inhibit T cell activation

Dasatinib (Dasatinib) as an inhibitor of Src/ABL kinase can effectively inhibit many functions of normal human T lymphocytes in vitro, including Dasatinib can effectively block TCR signaling transduction by binding LCK, inhibit T cell activation, cytokine secretion and proliferation in vitro, but does not affect T cell viability [ ¹ ]And dasatinib is known to inhibit TCR-mediated signal transduction, cell proliferation, cytokine secretion and cellular responses in vivo by inhibiting LCK phosphorylation. However, this inhibition is reversible and T cell function is restored when dasatinib is removed [ ² ]. Meanwhile, dasatinib is also a potential, rapid and reversible CAR-T cell function inhibitor, and can inhibit proliferation, cytokine secretion and in-vivo tumor killing activity of CAR-T cells [ ³ ]Can inhibit the secretion and proliferation of CAR-T cells in vivo and in vitro, andthis inhibition is reversible [ ⁴ ]。

The inventors have aimed at validating LCK ^T316I The mutation can make the universal CART cell resist the inhibition of the dasatinib to the activation of the T cell, and the dasatinib can effectively inhibit the TCR signal to inhibit the activation of the T cell is repeated.

The inventors treated T cells with dasatinib or DMSO for 5 hours in advance, then activated T cells with DynaBeads magnetic beads against CD3/CD28 for 24 hours, followed by flow detection of the expression of the activating molecules CD25, CD69 and 4-1 BB. As shown in fig. 13, DMSO control significantly upregulated the activation molecule CD25 and CD69 double positive cells from 2.85% to 38.9% after 24 hours of bead activation (fig. 13A), whereas the proportion of CD25 and CD69 double positive cells could not be upregulated after 24 hours of bead activation after 5 hours of treatment with 100nM or 1000nM dasatinib (fig. 13A, lower). Similarly, the DMSO control also significantly up-regulated the activation molecule CD25 and 4-1BB biscationic cells from 1.11% to 35.9% after 24 hours of bead activation (fig. 13B), whereas the CD25 and 4-1BB biscationic cell ratios could not be up-regulated again after 24 hours of bead activation after 5 hours of treatment with 100nM or 1000nM dasatinib (fig. 13B, bottom). These data suggest that dasatinib is able to inhibit activation of TCR signaling and that a concentration of 100nM is sufficient to have an inhibitory effect.

^T316I Example 4: LCK mutation renders T cells resistant to inhibition of TCR activation by dasatinib

To verify the tolerance of LCK-T316I mutations to dasatinib function, the inventors performed experimental verification of T cells prepared in example 2 that were LCK edited with different sgRNA lengths. Three different treatments were set for each group of electrotransformed T cells and for MockT cells not electrotransformed: as shown in fig. 14A, the first set of treatments was not treated with dasatinib and was not activated with CD3/CD28 magnetic beads (fig. 14A, first row); the second group was not treated with dasatinib, but TCR activation was performed for 24 hours with anti-CD 3/CD28 magnetic beads (fig. 14A, second row); the third group was pre-treated with 100nM dasatinib for 5 hours, followed by TCR activation with anti-CD 3/CD28 beads for 24hr (FIG. 14A, third row), and finally, LCK-T316I mutations were confirmed to be resistant to inhibition of T cell activation by dasatinib by flow-through detection of the expression of T cell activation signals such as CD25, CD69 and 4-1 BB.

T cells were edited and grouped as follows in this example: mockT group is a T cell that has not been electrotransformed, and group 01CBE3 is a T cell that has only been electrotransformed with CBE3 protein; 02-05 are T cells, respectively, which are electrotransformed with CBE3 protein and RNP formed by LCK-sgRNA12 of different lengths, because the sgRNA does not cause any amino acid mutation of LCK protein, and can be used as negative control for LCK editing; 06-08 are T cells, respectively, electrotransformed with CBE3 protein and RNP formed by LCK-sgRNA16 of different lengths, which have been mutated with LCK-T316I, the mutation efficiency being around 50%, see FIG. 11 of example 2.

The inventors demonstrated that LCK-T316I mutations can render T cells resistant to inhibition of TCR activation by dasatinib.

As shown in fig. 14, in the absence of dasatinib treatment and in the absence of anti-CD 3/CD28 magnetic bead activated T cells (fig. 14A, fig. 14B, first row), all T cells tested were devoid of expression of CD69 (fig. 14A) and 4-1BB (fig. 14B) activating molecules. When T cells were activated by the addition of anti-CD 3/CD28 beads (fig. 14A,14B, second row), expression of CD25, CD69 and 4-1BB was significantly increased in each group of cells, demonstrating that T cells were effectively activated by anti-CD 3/CD28 beads, transducing TCR signals, whether LCK was edited or not. After pretreatment with 100nM dasatinib for 7 hours, T cells were activated with anti-CD 3/CD28 beads (FIG. 14A, FIG. 14B, third row) when the expression of CD25, CD69 and 4-1BB activating molecules was consistent with the first row of unactivated groups in cells (MockT, 01-05) in which LCK was not edited except for T cells of LCK-T316I edited group (06-08), suggesting that 100nM dasatinib was able to completely inhibit TCR signaling and increased expression of CD69 and 4-1BB due to T cell activation. Whereas LCK-T316I mutated T cells (groups 06-08) were still stimulated by anti-CD 3/CD28 to activate and transduce TCR signals under 100nM dasatinib, exhibiting significantly increased expression of activating molecules such as CD25, CD69 and 4-1BB (FIGS. 14A,14B, third row 06-08), further demonstrating that LCK-T316I mutations can render T cells resistant to inhibition of TCR activation by dasatinib.

The inventors further performed statistics on the data, with MockT and 01-CBE3 groups set as blank control groups, 02-05 groups set as sgRNA12 edit group (synonymous mutant group), 06-08 groups set as sgRNA16 edit group (LCK-T316I mutant group), and the positive cell proportion expressing CD25, CD69, and 4-1BB, respectively, under three treatment conditions. As shown in fig. 14C, the expression ratio of the three activation markers was significantly increased in the sgRNA 16-edited group under the treatment with the addition of dasatinib and anti-CD 3/CD28 magnetic beads compared to the control group and the sgRNA 12-edited group. It was further suggested that T cells of the sgRNA 16-edited group (-50% LCK-T316I mutation) showed tolerance to dasatinib inhibiting the activation function of T cells, and that LCK-T316I mutated T cells were still able to be activated.

Furthermore, although the expression of the activated molecules was significantly reduced in the dasatinib treated group compared to the sgRNA16 group cells directly activated by anti-CD 3/CD28 magnetic beads without dasatinib treatment (around 20% -50%, fig. 14C). This is due to the fact that the efficiency of the sgRNA16 editing group is around 50% and 100% editing cannot be achieved, and that T cells with LCK-T316I mutations partially show tolerance under dasatinib treatment, while cells that have not been edited are still not activated effectively, eventually leading to a decrease in the activation rate. These data well illustrate the tolerability of LCK-T316I mutations to dasatinib to inhibit T cell activation function.

^T316I Example 5: LCK mutation makes anti-CD 19-CAR-T cells resistant to inhibition of CD107a transport by dasatinib

Based on the inventors' demonstration that LCK-T316I mutations can make T cells resistant to inhibition of TCR activation by dasatinib, in order to demonstrate that LCK-T316I mutations can make T cells resistant to inhibition of TCR activation by dasatinib, but also CAR-T cells resistant to inhibition of CD107a transport during CAR activation by dasatinib, the inventors have devised experiments demonstrating that LCK-T316I mutations make anti-CD 19-CAR-T cells resistant to inhibition of CD107a transport by dasatinib.

First, the inventors performed electrotransformation editing of the prepared anti-CD 19 CAR-T, and respectively electrotransformation of the CBE3 protein, the RNP complex of CBE3 and LCK-sgRNA12, and the RNP complex of CBE3 and LCK-sgRNA16, three different sets of editing treatments were performed, and after editing for 72 hours, mutation efficiencies were detected, in which LCK-T316I mutation reached about 43% (FIG. 15A). As shown in the protocol of fig. 15B, the inventors pre-treated each set of CAR-T cells for 12 hours with 100nM dasatinib, followed by co-incubation with different target cells (positive target cells Nalm6 expressing CD19 antigen and negative target cells K562 not expressing CD19 antigen) and anti-CD 19-CAR-T cells, stimulated activation of CAR signals, mediated CAR-T killing of target cells and CAR-T accumulation of CD107a. Monensin (monensin) and CD107a antibodies were added 5 hours prior to detection, and after 5 hours staining for CD8 and CAR positivity was performed for detection of enrichment of CD107a transport of CD8 positive CAR positive cells.

By analyzing the flow-through experimental data, the transport enrichment of CD107a was analyzed from CD8 and CAR biscationic cells as in the gate-on strategy shown in fig. 15C. As a result, it was found that, regardless of whether anti-CD 19-CAR-T cells were edited or not, in DMSO-treated control group, negative target cell K562 failed to activate CAR-T for efficient CD107a transport (first column from left in fig. 15D), whereas positive target cell Nalm6 was able to sufficiently activate CAR-T to release CD107a with a positive rate of 70% or more (third column from left in fig. 15D). In the 100nM dasatinib treated group, negative control group K562 showed little enrichment of CD107a transport (second column from left in FIG. 15D), whereas under positive target cell Nalm6 stimulation, only LCK-sgRNA16 edited group (LCK-T316I mutation ratio 43%) of CAR-T cells showed 50% positive CD107a transport (fourth column from left in FIG. 15D), suggesting that LCK-T316I mutation was indeed able to make anti-CD 19-CAR-T cells resistant to inhibition of CD107a transport by dasatinib.

The inventors performed a statistical analysis of two replicates of the above experiments, and as shown in fig. 15E, positive target cells Nalm6 were able to activate all anti-CD 19-CAR-T cells to release CD107a, and after 100nM dasatinib treatment, the CD107a release was significantly reduced in the CBE3 control group and LCK-sgRNA12 synonymous mutant group, while the CD107a release was still at a higher level in the LCK-sgRNA16-T316I mutant group, forming a significant difference from the control group. This phenomenon demonstrates that dasatinib can inhibit not only activation of TCR signaling in T cells but also CAR signaling in CAR-T cells, while LCK-T316I mutations can resist the inhibitory effect of dasatinib on CAR-T cell CAR signaling activation.

^T316I Example 6: LCK mutation makes anti-BCMA-CAR-T cells resistant to inhibition of CD107a transport by dasatinib

In example 5, the inventors have demonstrated that LCK-T316I mutation renders anti-CD 19-CAR-T cells resistant to inhibition of CD107a transport by dasatinib, and to strengthen this conclusion, in this example, the inventors have verified that LCK-T316I mutation still can be observed in CAR-T of BCMA target to render BCMA-CAR-T cells resistant to inhibition of CD107a transport by dasatinib.

First, the inventors performed electrotransformation editing of the prepared anti-BCMACAR-T, and respectively electrotransformation of CBE3 protein, RNP complex of CBE3 and LCK-sgRNA12, and RNP complex of CBE3 and LCK-sgRNA16, three different sets of editing treatments were realized, and after editing for 72 hours, mutation efficiencies were detected, in which LCK-T316I mutation reached about 42% (fig. 16A). The inventors pre-treated each group of CAR-T cells with 100nM dasatinib for 12 hours, followed by co-incubation with different target cells (positive target cells U266B1 and RPMI-8226 expressing BCMA antigen and negative target cells K562 not expressing BCMA antigen) and anti-BCMA-CAR-T cells, stimulated activation of CAR signals, mediated CAR-T killing of target cells and CAR-T accumulation of CD107a. The accumulation of CD107a transport for CD8 positive CAR positive cells was detected by adding monensin and CD107a antibodies 5 hours prior to detection, and staining for CD8 and CAR positive after 5 hours.

By analyzing the flow-through experimental data, the transport enrichment of CD107a was analyzed from CD8 and CAR biscationic cells. As a result, as shown in fig. 16B, in DMSO-treated control, negative target cells K562 failed to activate CAR-T for efficient CD107a transport (first column from left in fig. 16B), whereas positive target cells U266B1 and RMPI-8228 both fully activated CAR-T to release CD107a with a positive rate of 68% or more (third and fifth columns from left in fig. 16B), regardless of anti-BCMA-CAR-T cell editing or not. In the 100nM dasatinib treated group, the negative control group K562 showed little enrichment of CD107a transport (second column from left in FIG. 16B), whereas under stimulation of positive target cells U266B1 or RMPI-8226, only the CAR-T cells of the LCK-sgRNA16 edited group (LCK-T316I mutation ratio 42%) showed 39.6% and 44.7% positive CD107a transport (fourth and sixth columns from left in FIG. 16B), suggesting that the LCK-T316I mutation was indeed able to render anti-BCMA-CAR-T cells resistant to inhibition of CD107a transport by dasatinib.

The inventors performed a statistical analysis of two replicates of the above experiments, and as shown in fig. 16C, positive target cells U266B1 and RMPI-8228 were able to activate all anti-BCMA-CAR-T cells to release CD107a, and after 100nM dasatinib treatment, CD107a release was significantly reduced in CBE3 control group and LCK-sgRNA12 synonymous mutant group, while CD107a release was still at higher level in LCK-sgRNA16-T316I mutant group, forming a significant difference from control group. This result further demonstrates that LCK-T316I mutations are resistant to the inhibition of CAR-T cell CAR signal activation by dasatinib.

^T316I Example 7: LCK mutation to cause anti-CD 19/BCMA double-target UCAR-T cells to resist transformation of CD107a by dasatinib Inhibition of transport

In examples 5 and 6, the inventors have demonstrated that LCK-T316I mutations render anti-CD 19-CAR-T cells and anti-BCMA-CAR-T cells resistant to inhibition of CD107a transport by dasatinib, and to strengthen this conclusion, in this example, the inventors have further verified that inhibition of CD107a transport by dasatinib is still observed in CD19 and BCMA bispecific CAR-T cells.

The inventors performed electrotransformation editing on the prepared CD19/BCMA double-target CAR-T, and respectively electrotransformed the CBE3 protein, the CBE3 and the RNP complex (18 nt and 21 nt) of LCK-sgRNA12, and the CBE3 and the RNP complex (18 nt and 21 nt) of LCK-sgRNA16, thereby realizing three different editing processes. The inventors performed pretreatment of 100nM dasatinib for 12 hours on each set of CAR-T cells, followed by co-incubation with different target cells and anti-BCMA-CAR-T cells, verifying activation of CAR signal and release of CD107a by different target cells. These target cells include U266B1 (BCMA positive), raji (CD 19 positive), K562 (negative control), allogeneic T cells (negative control), and blank Buffer control (negative control). The accumulation of CD107a transport for CD8 positive CAR positive cells was detected by adding monensin and CD107a antibodies 5 hours prior to detection, and staining for CD8 and CAR positive after 5 hours.

Results as shown in figure 17A, transport enrichment of CD107A was analyzed from CD8 and CAR biscationic cells by analyzing flow experimental data. CD19/BCMA bispecific CAR-T cells that were not edited were able to be efficiently activated by U266B1 and Raji cells under DMSO treatment, CD107A expression was positive at 74.5% and 54.5%, but not activated by K562, host T, and Buffer (fig. 17A, first row). Under 100nM dasatinib treatment, LCK-T316I-edited CAR-T cells (FIG. 17A, second row to fifth row) were unable to be activated by both positive target cells U266B1 and Raij to release CD107A, and only LCK-T316I mutated CAR-T cells (FIG. 17A, sixth and seventh rows) were able to resist inhibition of CAR signal activation by dasatinib, whereas CD107A was released by U266B1 and Raji activation to a proportion of 50% positive or more. Fig. 17B is a bar graph illustration of 17A data. These data suggest that LCK-T316I mutations can render CD19/BCMA bispecific CAR-T cells resistant to inhibition of CD107a transport by dasatinib.

^T316I Example 8: LCK mutation makes anti-BCMA-CAR-T cells resistant to inhibition of CAR-T killing function by dasatinib

The foregoing example 4 demonstrates that mutation of LCK-T316I can render T cells resistant to inhibition of TCR activation by dasatinib, and examples 5-7 demonstrate that LCK-T316I mutation can render CAR-T cells resistant to inhibition of CAR activation and inhibition of CD107a transport by dasatinib. To further demonstrate the ability of LCK-T316I mutations to render CAR-T cells resistant to dasatinib to inhibit CAR-T killing, the inventors performed the following experiments on anti-BCMACAR-T cells.

The inventor experiment proves that the anti-BCMAAR-T after LCK-T316I mutation has tolerance to the CAR-T killing activity inhibition function of dasatinib.

The inventors performed experiments using the edited anti-BCMACAR-T cells in example 6, wherein LCK-T316I mutation efficiency was 42% (fig. 16A). As shown in fig. 18A, the inventors performed 12 hours pretreatment of 100nM dasatinib for each group of CAR-T cells, followed by and different luciferase-positive target cells (luciferase+) to effect target ratio 1:1 for 24 hours, two replicates were performed per group. After the incubation, the killing of the target cells by CAR-T cells was assessed by detecting luciferase activity, with lower luciferase numbers representing less target cells surviving and stronger CAR-T killing.

As shown in fig. 18B and 18C, in DMSO control, no matter whether LCK-T316 is edited or not, the anti-BCMA CAR-T can cause a significant decrease in the luciferase value of RPMI-8226 positive target cells without affecting the luciferase value of negative target cells Nalm6, demonstrating that the anti-BCMA CAR-T cells have a function of normally killing target cells. After stimulation of positive target cells RPMI-8226 with 100nM dasatinib, the luciferase values in CBE3 protein control and LCK unedited groups (cbe3+sg 12) were significantly higher than in DMSO groups, demonstrating that the killing function of anti-BCMA CAR-T was significantly inhibited with dasatinib treatment. While the luciferase values in LCK-T316I mutated CAR-T cells (CBE+sg 16) were significantly lower than in the other two groups, demonstrating that the anti-BCMA CAR-T after LCK-T316I mutation was resistant to the CART killing activity inhibitory function of dasatinib.

Repeated experiments by the inventor prove that the anti-BCMA CAR-T after LCK-T316I mutation has tolerance to CART killing activity inhibition function of dasatinib.

The inventors performed repeated killing experiments with and without editing of the LCK-edited anti-BCMA CAR-T cells described above. The experimental procedure is identical to that of fig. 18A in this example. Each group of CAR-T cells was pretreated with 100nM dasatinib for 12 hours, followed by and different luciferase-positive target cells at an effective target ratio of 1:1 for 24 hours, two replicates were performed per group. After incubation, the killing of the target cells by CAR-T cells was assessed by detecting luciferase activity, with lower luciferase values representing fewer surviving target cells and stronger CAR-T killing.

As shown in fig. 19A and 19B, in DMSO control, no matter whether LCK-T316 is edited or not, the anti-BCMA CAR-T can cause a significant decrease in the luciferase value of RPMI-8226 positive target cells without affecting the luciferase values of negative target cells K562 and Nalm6, demonstrating that the anti-BCMACAR-T cells have a function of normally killing the target cells. After stimulation of positive target cells RPMI-8226 with 100nM dasatinib, the luciferase values in CBE3 protein control and LCK unedited groups (cbe3+sg 12) were significantly higher than in DMSO groups, demonstrating that the killing function of anti-BCMA CAR-T was significantly inhibited with dasatinib treatment. Whereas the luciferase values in LCK-T316I mutated CAR-T cells (CBE+sg 16) were significantly lower than in the other two groups. Furthermore, while negative target cell K562 was partially activated for LCK-T316I-edited CAR-T cells under 100nM dasatinib treatment, negative target cell Raji did not activate anti-BCMA-CART cells, which was a better negative control. These data again demonstrate that anti-BCMA CAR-T after LCK-T316I mutation is resistant to the CART killing activity inhibitory function of dasatinib.

Example 9: dasatinib at 25nM concentration was effective in inhibiting T cell activation in vitro

In the above examples the inventors demonstrate that dasatinib at a concentration of 100nM is effective in inhibiting the activation of TCR and CAR signals, i.e. at this concentration the activation of normal T cells and CAR-T cells is inhibited. At this point, the LCK-T316I mutation can render T cells resistant to inhibition of activation by dasatinib, i.e., UCAR-T cells having undergone the LCK-T316I mutation can retain the ability to be activated by and kill target cells in a 100nM concentration of dasatinib environment.

Based on this, the strategy proposed by this patent to achieve universal CAR-T is to pre-treat patients with dasatinib, inhibit the activation function of host T cells, and then reinject LCK-T316I mutated UCAR-T cells. In this case, host T cells fail to clear UCAR-T cells due to inhibition of TCR activation by dasatinib; meanwhile, UCAR-T can resist the inhibition of the dasatinib on the activation of CAR signals due to carrying LCK-T316I mutation, and target malignant tumor cells to mediate tumor elimination.

The inventors have demonstrated in example 3 that 100nM dasatinib is sufficient to inhibit activation of T cell TCR signaling. To further explore the threshold concentration of dasatinib for T cell activation inhibition and thus guide clinical medication, the inventors further explored lower concentrations of dasatinib.

The inventors isolated CD3 positive T cells from human peripheral blood and treated them in eleven aliquots of 1e5 cells each. 1 part of the samples were not treated with dasatinib, nor were anti-CD 3/CD28 DynaBeads added to activate TCR as initial control; an additional 10 cells were pre-treated with dasatinib at different concentrations for 4 hours, setting ten concentrations in the interval from 0nM to 50nM, followed by addition of anti-CD 3/CD28 DynaBeads to activate TCR signals overnight. The next day the expression of CD25, CD69 and 4-1BB activation signals was examined, and TCR signal activation was examined. Two replicates were set for each set of experiments.

As shown in FIG. 20A, after T cells not treated with dasatinib were activated by anti-CD 3/CD28 beads, a 60.7% proportion of CD25/CD69 double positive cells were activated (FIG. 20A first row, second row) demonstrating that anti-CD 3/CD28 beads were able to effectively activate the batch of T cells. Activation of TCR signals by anti-CD 3/CD28 magnetic beads was gradually attenuated by the gradient addition of 10-50nM concentration of dasatinib (fig. 20A first row, third column to sixth column, second row, first column to fifth column). The proportion of CD25 and CD69 double positive cells gradually decreased from 60.7% to 8.75% in the dasatinib treatment interval from 10nM to 20nM, exhibiting a pronounced dose-dependent effect. While the proportion of CD25 and CD69 double positive cells was significantly reduced to 1.06% at 25nM dasatinib, the proportion of double positive cells remained substantially below 1% after the concentration continued to rise to 50 nM.

Meanwhile, as shown in FIG. 20B, CD25 and 4-1BB were used as indicators of activated T cells, and a conclusion consistent with FIG. 11A was reached. Activation of TCR signals by anti-CD 3/CD28 magnetic beads was gradually attenuated by gradient addition of 10-50nM concentration dasatinib (fig. 20B first row third column to sixth column, second row first column to fifth column). The proportion of CD25 and 4-1BB biscationic cells gradually decreased from 31.0% to 2.09% in the dasatinib treatment interval from 10nM to 20nM, exhibiting a pronounced dose-dependent effect. While the proportion of CD25 and CD69 double positive cells was significantly reduced to 0.26% at 25nM dasatinib, the proportion of double positive cells remained substantially below 1% after the concentration continued to rise to 50 nM.

The above data demonstrate that dasatinib at a concentration of 25nM is effective in inhibiting T cell activation function in vitro. Basically can guide the minimum in vivo concentration of dasatinib required to achieve the purpose of inhibiting the activation of TCR signals in clinical medication to be 25nM, and can inhibit the activation of host T cells when the in vivo concentration of dasatinib is presumed to be above 25 nM.

Example 10: there is a continuing effect of dasatinib on the inhibition of T cell activation function

In example 9, the inventors demonstrated that dasatinib at a concentration of 25nM was effective in inhibiting T cell activation function in vitro, thereby directing the in vivo drug concentration. According to the existing dasatinib pharmacokinetic data [ ¹² ]The inventors found that dasatinib has a very short half-life in vivo, 3-4 hours, compared to other tyrosine kinase inhibitors. At the same time, for CML and ALL patients with different courses, the dose of dasatinib is 100mg or 140mg daily, or twice a day, and the dose is halved. Thus, dasatinib is metabolized faster in vivo and is most likely present at concentrations below 25nM for several hours in the dosing interval. In order to fully consider the factor of the in-vivo dasatinib concentration fluctuation, the inventor designs an in-vitro dasatinib dynamic concentration experiment to detect whether the inhibition of T cell activation function is still effective under the dynamic concentration change.

In this example, CD5-UCAR-T cells were used with double knockouts of CD5 and TRAC, and were edited by CBE3+LCK-sgRNA16 or CBE3+sgRNA12 to effect preparation of LCK-T316I mutant and control UCAR-T cells. Subsequently, treatment was carried out overnight with 50nM or 25nM concentrations, respectively. The next day the concentration of dasatinib was reduced as shown in figure 21a, adjusted to 0, 15, 25, 35 and 50nM after 50nM pretreatment overnight, respectively. Concentrations were adjusted to 0, 10, 15, 20 and 25nM, respectively, after overnight pretreatment as in FIG. 21B,25 nM. Meanwhile, jurkat cells are used as positive target cells for activation, and the target ratio is 1:1, simultaneously adding CD107a and monensin. After 4 hours of co-incubation, CD107a release was detected in CD8 and CAR biscationic cells.

The results show that the sg12 control CD5-UCART cells with LCK synonymous mutations reduced in concentration to 0nM after 50nM dasatinib pretreatment, which resulted in 12% positive CD107a release after 4 hours of activation by Jurkat target cells (first row of fig. 21A), and none of CD107a release after concentration reduction to 15, 25, 35, 50nM (second to fifth row of fig. 21A), demonstrated that a change in dasatinib concentration to 15nM after 50nM pretreatment was sufficient to inhibit UCAR-T cell activation within 4 hours. In addition, the sg16 edited group CD5-UCART cells with LCK-T316I mutation reduced to 0nM after 50nM dasatinib pretreatment, which resulted in 16% positive CD107a release after 4 hours of activation by Jurkat target cells (second row first column in fig. 21A), and still higher levels of CD107a release after concentration reduction to 15, 25, 35, 50nM (second row second to fifth column in fig. 21A), demonstrated that LCK-T316I mutation was able to tolerate inhibition of CAR signal by dasatinib.

Consistent with the results of 50nM dasatinib pretreatment experiments, the inventors demonstrated that adjusting the concentration to 10nM after 25nM dasatinib pretreatment was sufficient to inhibit activation of control CD5-UCAR-T cells by target cells to release CD107a (first row, second row of FIG. 21B), while the sg16 edited CD5-UCART cells with LCK-T316I mutation had a higher level of CD107a release after decreasing dasatinib concentration (second row, fifth row of FIG. 21B), again demonstrating that LCK-T316I mutation was able to tolerate inhibition of CAR signal by dasatinib.

The above experiments demonstrate that dasatinib has a continuing effect on the inhibition of T cell activation function. Dasatinib reduced from 50nM to 15nM or from 25nM to 10nM at dynamic concentration changes in vitro can inhibit T cell activation function, while LCK-T316I mutations can be resistant to such inhibition.

To repeatedly verify that there is a continued effect of dasatinib on the inhibition of T cell activation function, the inventors repeated the above experiments. CD5-CAR-T cells were pre-treated with 50nM or 25nM dasatinib overnight, the next day the drug concentration was adjusted down and incubated with positive or negative target cells while adding monensin blocker and CD107a antibody, and after incubation for 4 hours CD107a release from CD8/CAR biscationic cells was detected.

As shown in fig. 22A, CD5-CART cells were activated by Jurkat in the dasatinib-free environment with 23.6% CD107a positive cells (first row of first column in fig. 22A), reduced in concentration to 0nM after 50nM dasatinib pretreatment, which resulted in 12.1% positive CD107a release after 4 hours of activation by Jurkat target cells (second row of first column in fig. 22A), none of which had significant CD107a release after concentration reduction to 10, 20, 30, 40 and 50nM (third to seventh rows of first row in fig. 13A), demonstrating that changing dasatinib concentration to 10nM-20nM after 50nM pretreatment was sufficient to inhibit CAR-T cell activation within 4 hours. Meanwhile, neither CD5 knockdown CCRF cells as negative target cells nor Buffer negative control group were able to effectively activate CD5-CAR-T cells to release CD107a (fig. 22A, second and third rows).

As shown in fig. 22B, consistent with the results of 50nM dasatinib pretreatment experiments, the inventors demonstrated that adjusting the concentration to 10nM after 25nM dasatinib pretreatment was sufficient to inhibit activation of CD5-CAR-T cells by positive Jurkat target cells to release CD107a (fig. 22B, first row, third column).

Taken together, the two experiments described above essentially demonstrate that dasatinib has a continuing effect on the inhibition of T cell activation function. Dasatinib reduced from 50nM to 10-15nM or from 25nM to 10nM can inhibit T cell activation while LCK-T316I mutation can resist such inhibition under dynamic concentration changes in vitro.

The anti-CD 5-CAR molecules used herein are identical in sequence to those used in the examples that follow, and are CAR molecules that are related to a single domain antibody sequence, the specific sequences of which are set forth in SEQ ID NOs:57-76. The overall structure of this CAR is a second generation CAR molecule consisting of two single domain heavy chains against CD5 (SEQ ID NOs: 57-60), a junction region (SEQ ID NOs: 61-62), a CD8a region (SEQ ID NOs: 63-64), a CD28 intracellular domain (SEQ ID NOs: 65-66), a CD3z intracellular signaling domain (SEQ ID NOs: 67-68), a T2A sequence (SEQ ID NOs: 69-70), a CSF2RA signal (SEQ ID NOs: 71-72), and a tEGFR signal (SEQ ID NOs: 73-74). The total sequence of the final anti-CD 19-CD28z-CAR signal is set forth in SEQ ID NOs:75-76.

Example 11: verification that dasatinib can effectively inhibit NK cell activation

Dasatinib has inhibition function on activation of T cells and CAR-T cells, and can inhibit human beingsDegranulation and cytokine Release of Primary NK cells [ ⁵ ]. Whereas clearance by functional host T and NK cells is the main reason why UCAR-T is difficult to amplify in a host, in this invention, the treatment of dasatinib in UCAR-T treatment can enhance the survival of UCAR-T cells in a host not only by inhibiting activation of host T cells but also by inhibiting activation of host NK cells.

The inventors first verified that dasatinib was able to effectively inhibit NK cell activation.

The inventor uses Meitian and gentle human NK sorting magnetic beads to separate NK cells from human peripheral blood, and uses Davidae as NK culture medium to culture and amplify the NK cells in vitro, so that more than 95% of NK cell populations positive for CD56 expression can be obtained on the eighth day. This fraction of NK cells was removed, treated with dasatinib at 0nM, 100nM and 200nM, respectively, for 24 hours, followed by division into two groups, one of which was not subjected to any activation, and the other group was stimulated with MHC-class I molecule-deficient K562 cells for 5 hours, followed by co-detection of NK cell CD107a release.

As shown in fig. 23, the transport of CD107a was 11.4% in the non-activated group of NK cells without dasatinib (first row, third column) and the basal CD107a transport was reduced to 2% baseline after dasatinib treatment (first row, second column). When K562 target cells were added to activate NK cells, the untreated group CD107a transport was activated to 64.5% (second row, third column), whereas the dasatinib treated group CD107a transport was reduced to 2% baseline (second row, first, second column), suggesting that 100nM dasatinib was sufficient to inhibit NK cell CD107a transport, inhibiting NK cell activation.

Example 12: dasatinib at 30nM concentration was effective in inhibiting NK cell activation in vitro

In the above examples the inventors demonstrate that dasatinib at a concentration of 100nM is effective in inhibiting NK cell activation. In order to further explore the concentration threshold of dasatinib for NK cell activation inhibition and further guide clinical medication, the inventors further explored lower concentrations of dasatinib.

The inventor separates NK cells from human PBMC, and cultures and differentiates the NK cells by using an NK culture medium to obtain the CD56 positive NK cells with the purity higher than 95 percent. The search for NK activation threshold values was performed with seven concentrations of dasatinib in the range of 0-50nM, 0, 10, 20, 25, 30, 40 and 50nM, respectively, and treated overnight. The next day was resuspended in NK medium and K562 was added for activation, with lymphocyte activator as positive control for activation, also retaining a portion of NK cells not activated by K562, and each group was set up in duplicate. CD107a antibody and monensin were added simultaneously with activation, and CD107a release from CD56 positive NK cells was flow-detected after 6 hours of co-incubation.

As shown in fig. 24A, NK cells with lymphocyte activators added had a CD107a transport of 80% or more (first row in fig. 24A) and a K562 activation of 76% or more (third row in fig. 24) without dasatinib treatment. As the concentration of dasatinib increases, the proportion of NK cells that are activated by K562 to express CD107a gradually decreases. When dasatinib concentration reached 30nM, K562 was substantially unable to activate NK cells causing them to release CD107a (fig. 24A, second row below), demonstrating that dasatinib concentration of 30nM was able to effectively exert inhibition of NK cell activation function in vitro. As shown in fig. 24B, the inventors performed a significance analysis of the above experimental data in a bar graph, basically determining that a concentration of 30nM can significantly inhibit NK cell activation compared to a lower dasatinib concentration.

The data can basically guide the clinical application to achieve the minimum in-vivo concentration (30 nM) of dasatinib for inhibiting NK cells, and can inhibit the activation of host NK cells when the in-vivo concentration of dasatinib is presumed to be above 30 nM.

Example 13: in vitro inhibition of NK cell activation function by dasatinib has a continuous effect

In example 12, the inventors demonstrated that dasatinib at a concentration of 30nM was effective in inhibiting NK cell activation function in vitro, thereby guiding in vivo drug concentration. It has been mentioned in example 10 that dasatinib has a very short half-life in vivo, is metabolized faster in vivo, and is highly likely to be present for several hours in the dosing interval, compared to other tyrosine kinase inhibitors The concentration of the surfactant is less than 30nM ¹² ]. In order to fully consider the factor of the in-vivo dasatinib concentration fluctuation, the inventor designs an in-vitro dasatinib dynamic concentration experiment to detect whether the inhibition of NK cell activation function is still effective under the dynamic concentration change.

The inventor separates NK cells from human PBMC, and cultures and differentiates the NK cells by using an NK culture medium to obtain the CD56 positive NK cells with the purity higher than 95 percent. Respectively performing three kinds of pretreatment (1) without adding dasatinib pretreatment; (2) pretreatment with 25nM dasatinib; (3) pretreatment with 50nM dasatinib and incubation overnight. The next day the concentration of dasatinib was reduced and adjusted to 0, 7.5, 12.5, 17.5 and 25nM, respectively. Simultaneously, K562 cells are used as positive target cells to activate NK cells according to the effective target ratio of 1:1, simultaneously adding CD107a and monensin. Two replicates were set for each group. After 5 hours of co-incubation, CD107a release was detected in CD56 positive NK cells.

As shown in fig. 25A, NK cells were significantly activated by K562 after pretreatment with three different concentrations of dasatinib and a further decrease in concentration to 0nM, with CD107a release all above 58% (second column in fig. 25). At reduced concentrations to 7.5nM, NK cells of the 25nM and 50nM dasatinib pre-treated groups were not substantially activated by K562, and CD107a release was less than 1% (fourth column of FIG. 25). When the concentration was changed to 12.5, 17.5 and 25nM the next day, NK cells of 25nM and 50nM dasatinib pre-treated groups were more unable to be activated by K562, and the release of CD107a was less than 1% (sixth, eighth and tenth columns of FIG. 25).

As shown in fig. 25B, the inventors performed a significance analysis of the above experimental data to further demonstrate that NK cells were reduced to 7.5nM for 4 hours after pretreatment with 25nM or 50nM dasatinib and failed to be effectively activated by K562 cells, whereas cells without pretreatment were directly treated with 7.5nM dasatinib for 4 hours, and still had about 4.4% of the cells activated, with significant differences. Thus, the above experiments demonstrate that there is a continued effect of dasatinib on the inhibition of NK cell activation function. Dasatinib reduced from 25nM or 50nM post pretreatment to 7.5nM had substantially inhibited NK cell activation at dynamic concentration changes in vitro.

The above experiments demonstrate that dasatinib has a continuing effect on the inhibition of NK cell activation function. Dasatinib reduced from 25nM or 50nM pretreatment to 7.5nM has been shown to inhibit NK cell activation function under dynamic concentration changes in vitro.

To repeatedly verify that there is a continued effect of dasatinib on the inhibition of NK cell activation function, the inventors repeated the above experiments. NK cells were pre-treated with 50nM, 25nM and 10nM dasatinib overnight, the next day the drug concentration was adjusted down and incubated with positive K562 or negative Buffer with the addition of monensin blocker and CD107a antibody and after 4 hours incubation CD107a release from CD56 positive NK cells was detected.

As shown in fig. 26A, NK cells activated by K562 in the absence of dasatinib (first row of first column in fig. 26A) had 61.5% CD107a positive cells, decreased in concentration to 0nM after 50nM dasatinib pretreatment, which resulted in 39.7% positive CD107a release after 4 hours of activation by K562 target cells (second row of first column in fig. 26A), none of which had significant CD107a release after concentration decreases to 10, 20, 30, 40 and 50nM (third to seventh rows of first row in fig. 26A), demonstrating that a change in dasatinib concentration to 10nM after 50nM pretreatment was sufficient to inhibit NK cell activation within 4 hours. Meanwhile, the Buffer negative control group failed to effectively activate NK cells to release CD107A (fig. 17A second row).

As shown in fig. 26B, consistent with the results of 50nM dasatinib pretreatment experiments, the inventors demonstrated that adjusting the concentrations to 10nM after 10nM and 25nM dasatinib pretreatment was also sufficient to inhibit NK cell activation by positive K562 target cells and release CD107a (first row, third, fifth columns of fig. 26B). 10nM in this experiment substantially completely inhibited NK activation.

Taken together, the two experiments described above, basically demonstrate that there is a continuing effect of dasatinib on the inhibition of NK cell activation function. Dasatinib can inhibit NK cell activation function under dynamic concentration change in vitro from 50nM to 10nM or from 25nM to 10 nM.

^T316I Example 14: LCK mutation and dasatinib combination strategy allows for in vitro targeting of CD5-UCART at 5 to 1 effective target rates Mixed showerHas the advantages of amplification and persistence in the barcell reaction

The prepared CD5-UCART and BCMA-UCART with sufficient quantity are utilized to carry out in vitro mixed lymphocyte reaction, so that whether the combination of in vivo LCK-T316I-UCART (Km-UCART) and dasatinib can obtain survival space and even proliferation advantage in the host T environment is simulated.

The present invention relates to the knockout of the TRAC gene to produce universal CAR-T cells. Given that CD5 antigen is expressed on chronic B-lymphocyte leukemia cells, the universal CAR-T cells referred to in the following embodiments target CD5. However, it is known that CD5 is also expressed on all peripheral blood T cells and a very small portion of B cells, and that UCART cells targeting CD5 will recognize and kill autologous or allogeneic T cells. Thus, the present invention uses gene editing technology to knock out the CD5 gene and the TRAC gene simultaneously in T cells to prepare CD5-UCART cells.

CD5-UCART cells were prepared in smaller numbers from healthy donors, and in cell samples prepared in this batch, the inventors measured the knockout efficiency of TRAC gene by flow cytometry means >90%; the knockout efficiency of the CD5 gene is >99%; the T cells expressing CAR were measured to be approximately 35% by detection of EGFR transduction markers. The inventors performed further gene editing on the CD5-UCART cells successfully constructed above for in vitro testing, this gene editing using a CBE3 base editor aimed at creating T316I mutation at LCK site on CD5-UCART cells: the CBE3 group is CD5-UCAT cell control group; the sg12 group is a CD5-UCAT cell control group with synonymous mutation aiming at LCK; the sg16 group (Km group) was a CD5-UCAT cell experimental group in which LCK-T316I editing occurred.

The present invention contemplates that UCART cells are subjected to LCK-T316I to construct UCART that is resistant to dasatinib inhibition, and that in the host T cell environment in which dasatinib is present, host T cells are (almost) fully inhibited from killing by genetic engineering that has not been subjected to dasatinib inhibition, at which time UCART of the present invention is able to be released from immune rejection that would otherwise be mediated by host T cells to maintain normal proliferation rates.

The inventors used a Mixed Lymphocyte Reaction (MLR) model to assess the ability of Km-CD5-UCART cells to resist T cell mediated immune rejection in vitro and to clear allogeneic T cells (host T cells are also target cells for CD5-UCART cells since there is no knockout of the endogenous CD5 gene).

The method comprises the following steps:

(1) Sorting and activation of cd3+ T cells;

(2) CRISPR-Cas9 and sgRNA mediated TRAC, CD5 gene knockout;

(3) Lentivirus infects TRAC/CD5 knocked-out T cells;

(4) TRAC, CD5 gene knockout efficiency detection: as shown in fig. 27A, the knockout efficiency of the TRAC gene is >90% and the knockout efficiency of the CD5 gene is >99%;

(5) CAR positive rate detection: the CAR positive rate was measured by flow cytometry 5 days after infection of the T cells with lentiviruses, and the ratio of CAR expressing T cells was about 35% to 40% measured with two different markers as shown in fig. 27B. At that time, the inventors considered that CD5-UCART cells were obtained that could be used for in vitro testing;

(6) T316I editing (Km) of CBE3 and sgRNA mediated LCK genes;

(7) Efficiency detection of Km editing by CD5-UCART cells: as a result, as shown in FIG. 27C, km editing did not occur in both CBE3 group and sg12 group; the mutation efficiency of T316I in the sg16 (Km) group CD5-UCART cells is 79%, i.e. 79% of CD5-UCART cells can theoretically resist the inhibition effect of dasatinib;

(8) Obtaining simulated host T cells in vitro experiments: in order to test the function of Km-CD5-UCART in allogeneic T cell environments, the inventors have further required to obtain donor-derived T cells, as allogeneic host T cells, that are distinct from the HLA-ABC protein of the donor involved in the preparation of Km-CD 5-UCART. The freshly isolated allogeneic host T cells were not activated with anti-CD 3/-CD28 antibody magnetic beads and were pretreated with 100nM dasatinib, i.e., dasatinib was added to the T cell complete medium before the assay began. Such treatment is intended to mimic the need to administer dasatinib drug pretreatment to patients to inhibit host T cell mediated effector function prior to use of Km-UCART as a therapeutic drug;

(9) The survival of Km-CD5-UCART cells in the host T cell environment was examined using mixed lymphocyte reaction: this mixed lymphocyte reaction was aimed at examining the ability of Km-CD5-UCART cells to resist T cell mediated immune rejection in vitro and to clear allogeneic T cells, so the inventors considered Km-CD5-UCART as a theoretical effector cell and allogeneic host T cells as target cells.

Effector cells in this mixed lymphocyte reaction were divided into 3 groups: (1) the CBE3 group is a CD5-UCAT cell control group; (2) The sg12 group is a CD5-UCAT cell control group with synonymous mutation aiming at LCK; (3) The sg16 group (Km group) was a CD5-UCAT cell experimental group in which LCKT316I editing had occurred. The target cells in this mixed lymphocyte reaction only use allogeneic T cells from the same donor source. Dasatinib was added to T cell complete medium before the start of the MLR assay, i.e. pretreatment of the simulated host T cells (target cells) and CD5-UCART cells (effector cells) was performed for 12 hours, respectively.

This embodiment is described in 1: 5-effect target ratio development it is stated here that the inventors defined the effect target ratio as the absolute number of cells of CD5-UCART expressing CAR molecules (35% CAR positive rate in this example) versus the absolute number of mock host T cells. The effector cells of 3 groups were then cultured according to 8.09×10 ⁴ 、5.20×10 ⁴ 、1.26×10 ⁴ The number of cells was two wells each in a flat bottom 96-well plate (100. Mu.l volume), and the target cells were plated 1.42X 10 each ⁵ 、9.10×10 ⁴ 、2.20×10 ⁴ The number of individual cells (100. Mu.L volume) was mixed with effector cells and the total volume per well was 200. Mu.L, all cultured in complete medium for T cells. In the prior 3 groups of mixed wells of effector cells and target cells, 100nM dasatinib is added to one of the wells serving as an experimental well, and the same volume of DMSO is added to the other well serving as a control well (thus, in the embodiment, the dasatinib drug solvent is DMSO). The well plate was placed in an incubator at 37℃for cultivation.

This test, the inventors predicted that sg16 effector cells could exhibit growth advantages over other groups in dasatinib-treated groups. The inventors thus need to collect the following data during the course of the mixed lymphocyte reaction: cell counts at different time points; ratio and absolute number of effector cells to target cells at different time points; CAR positive rate of effector cells.

To obtain the above data, the inventors have taken a part of cells from each well on day 2 (day 0 of plating) of co-culture to count cells, and the ratio of the total cells in each well to the total cells in each well is determined, and the absolute number of cells in each well is calculated from the ratio and the count result of the taken cells. The removed cells also need to be used in flow cytometric analysis to determine the ratio of effector cells, target cells, and CAR positive rate of effector cells.

The inventors calculated the index of interest by combining the cell count results at each time point with the flow cytometric analysis results, and FIG. 28A shows the percentage relationship of UCAR-T in the MLR overall system. In the MLR reaction, UCART cells in a DMSO group can kill the host T cells, and the host T cells are reduced to below 5% on the 10 th day of the test; under 100nM dasatinib treatment, the UCAR-T of the control group is inhibited against the host T cells, the proliferation of the host T is obvious, only the UCAR-T edited by Km can resist the inhibition of dasatinib, and the UCAR-T killing function under normal conditions is restored.

For the variation of host-T in the total system, as shown in FIG. 28B, UCAR-T in the DMSO group can kill the host T cells, and the host T cells of D10 are reduced to below 5%; under 100nM dasatinib treatment, the UCAR-T of the control group is inhibited against the host T cells, the proliferation of the host T cells is obvious, only the UCAR-T edited by Km can resist the inhibition of dasatinib, and the UCAR-T killing function under normal conditions is restored.

Meanwhile, as shown in fig. 28C, the CAR positive rate of each group gradually increased to 70% with the increase of the number of MLR days, but did not show the inter-group difference, and in this test, the inventors thought that the functional component, i.e., the CAR positive cells, did not obtain faster enrichment due to Km editing in combination with dasatinib than the control group. In this test, the real aging target ratio was favorable to reflect the dynamic relationship between the effector components in the system and the mock host T cells, as shown in fig. 28D, the Km-CD5-UCART group under dasatinib treatment exhibited a sustained increase in the effective target ratio, whereas the other control groups did not. Thus, the inventors demonstrated that dasatinib was able to inhibit the clearance of CD5-UCART from host T cells, whereas KT316I mutations were able to effectively restore UCAR-T function.

^T316I Example 15: LCK mutation and dasatinib combined strategy enables effective target ratio of CD5-UCART at 50-100 to 1 Has the advantages of amplification and persistence in vitro mixed lymphocyte reaction

This example is a supplement and optimization of example 14, and aims to evaluate the ability of Km-CD5-UCART cells to resist in vitro T cell mediated immune rejection and to clear allogeneic T cells by a Mixed Lymphocyte Reaction (MLR) model at more extreme allogeneic T numbers.

The method comprises the following steps:

(1) Sorting of cd3+ T cells;

(2) TRAC, CD5 gene knockout, T316I editing (Km) of LCK gene are carried out on the T cells which are not activated;

(3) Activating T cells after electroporation;

(4) Lentivirus infects T cells;

(5) TRAC, CD5 gene knockout efficiency, the result is shown in FIG. 29A, the TRAC gene knockout efficiency >95%, and the CD5 gene knockout efficiency >99.5%.

(6) CAR positive rate detection, results shown in fig. 29B, was about 30%;

(7) Km editing efficiency was detected, and as a result, as shown in fig. 29C, km editing did not occur in Ctrl group; the mutation efficiency of T316I in CD5-UCART cells in Km group was 45%. At that time, the inventors considered that Km-CD5-UCART cells were obtained that could be used for in vitro testing;

(8) Obtaining simulated host T cells in vitro experiments: in order to test the function of Km-CD5-UCART in the environment of host T cells, the inventors have further required to obtain donor-derived T cells as host T cells that are different from the HLA-ABC protein of the donor involved in preparing Km-CD 5-UCART. The batch of T cells was obtained as described in step (1) of the present example, and the cell purity was also examined by flow cytometry. The freshly isolated host T cells were not activated with anti-CD 3/-CD28 antibody magnetic beads and were pretreated with 100nM dasatinib, i.e. dasatinib was added to the T cell complete medium before the assay began. Such treatment is intended to mimic the administration of dasatinib drug pretreatment to patients to inhibit host T cell mediated effector function prior to the administration of Km-UCART as a therapeutic drug.

(9) The survival of Km-CD5-UCART cells in the host T cell environment was examined using mixed lymphocyte reaction: this mixed lymphocyte reaction was intended to examine the ability of Km-CD5-UCART cells to resist T cell mediated immune rejection in vitro and to clear T cells from the same host, so the inventors considered Km-CD5-UCART as a theoretical effector cell and host T cells from the host as target cells.

Effector cells in this mixed lymphocyte reaction were divided into 2 groups: (1) The Ctrl group is a CD5-UCAT cell control group with synonymous mutation aiming at LCK; (2) Km groups are CD5-UCAT cell experimental groups in which LCKT316I editing occurred. The target cells in this mixed lymphocyte reaction only use the same donor-derived, homogeneous host T cells. Dasatinib was added to T cell complete medium before the start of the MLR assay, i.e. pretreatment of the simulated host T cells (target cells) and CD5-UCART cells (effector cells) was performed for 24 hours, respectively.

The present embodiment is intended to be 1: 50-100 effective target ratio. The effector cells of group 2 were treated with 2.5X10 ⁵ The number of cells was 6 wells each in a flat bottom 96-well plate (250. Mu.l volume) and the target cells were plated 1.5X10 respectively ⁶ The number of individual cells (250. Mu.l volume) was mixed with effector cells and the total volume per well was 500. Mu.l, all cultured in complete medium for T cells. The existing 2 groups of mixed wells of effector cells and target cells were each given 3 treatments, each setting 2 technical replicates: treatment one was DMSO, treatment two was 25nM dasatinib, and treatment three was 50nM dasatinib. The well plate was placed in an incubator at 37℃for cultivation. Dasatinib and DMSO in the culture system were supplemented every two days.

This test, the inventors predicted that Km group effector cells could exhibit growth advantages over other groups in dasatinib-treated groups, and based on the data already obtained in the above examples, the inventors speculated that Km group treated with 50nM dasatinib had a growth advantage over Km group effector cells treated with 25nM dasatinib, i.e. that Km-CD5-UCART achieved growth advantages under dasatinib treatment with dose-dependent effects on dasatinib. To verify the above hypothesis, the inventors need to collect the following data during the course of the mixed lymphocyte reaction: cell counts at different time points; ratio and absolute number of effector cells to target cells at different time points; CAR positive rate of effector cells.

To obtain the above data, the inventors have taken a part of cells from each well on

days

2, 5, 8, 11, and 13 of co-culture (day of plating is day 0) to count the cells, and the ratio of the total cells in each well to the cells to be taken out is determined, and the absolute number of cells in each well is calculated from the ratio and the count result of the cells to be taken out. The removed cells also need to be used in flow cytometric analysis to determine the ratio of effector cells, target cells, and CAR positive rate of effector cells.

The inventors in this mixed lymphocyte reaction calculated the index of interest by combining the cell count results at each time point with the flow cytometry results. The actual plating cell number on day 0 was obtained by cell counting and flow cytometry after plating, without regard to the amount of cells to be added at the time of experimental design. Because errors in loading can lead to inconsistent actual plating and cell volume for the planned inoculum, and secondly because of the set up of technical replicates, counting the actual inoculum volume makes the results easier to carry out for the biological statistical analysis. The actual CAR positive rate of the effector cells is about 10% in the test, and the actual target ratio obtained by combining the cell counting result and the flow cytometry analysis result is 1:50-1:100. After the end of this test, the inventors performed detailed statistical analysis of the dynamic change in the number of effector cells (fig. 30A), the dynamic change in the number of target cells (fig. 30C), and the CAR positive rate in effector cells (fig. 30E), respectively, based on the various indexes obtained at each time point.

As the data in fig. 30A show, neither Ctrl-CD5-UCART nor Km-CD5-UCART can be effectively expanded in the environment of host T cells under DMSO treatment, suggesting that host T cells are activated by UCART cells and perform their killing effect, such that UCART cells are gradually cleared. Under 25nM dasatinib treatment, the proliferation of Ctrl-CD5-UCART of the control group is inhibited, and Km-CD5-UCART can be effectively amplified, which suggests that the function of the host T cells is inhibited by dasatinib, and normal proliferation can be obtained and the host T cells can be effectively killed due to the inhibition of the resistance of Km-CD5-UCART to dasatinib. The trend of 50nM dasatinib treatment was consistent with 25nM treatment, and by day 11 of the test, by the time of 25nM treatment, the Km-CD5-UCART under 50nM dasatinib treatment gave a more pronounced sustained proliferation advantage over 25nM treatment, suggesting that 50nM inhibition of activation of host T cells was more effective, thereby more significantly reducing the survival pressure of Km-CD5-UCART in the host T cell environment.

In addition, from the analysis of the proportion of total cells occupied by effector cells, as shown in fig. 30B, the proportion of Km-CD5-UCART culture system under dasatinib treatment is significantly increased over time, while the other groups are opposite to each other, suggesting that Km-CD5-UCART can tolerate the activation inhibition of dasatinib, still effectively expand in high-fold host T cell environment, and that proliferation advantage of Km-CD5-UCART is more sustainable under 50nM dasatinib treatment than under 25nM dasatinib treatment. These data confirm the inventors' hypothesis that the growth advantage obtained by Km-CD5-UCART under dasatinib treatment has a dose dependent effect on dasatinib.

For the dynamic changes of host T cells, as shown in fig. 30C, the numbers of host T cells in each group were all shown to be dynamic, and no different rule of change between groups was shown. However, it was not difficult to find from the analysis of the host T cell ratio (FIG. 30D), that by the 11 th day of the test, the host T cell ratio of the Km-CD5-UCART group under dasatinib treatment was continuously decreased, while the host T cell ratio of the control group remained stable for a long period. The inventors analyzed that the killing function was (almost) completely inhibited due to the host T cells not receiving the genetic engineering modification to tolerate dasatinib inhibition, and Km-CD5-UCART became the main functional component in the culture system due to the inhibition of dasatinib, which could continuously clear the host T cells, resulting in a continuously decreasing proportion thereof. This result demonstrates the inventor's hypothesis from another point of view that Km-CD5-UCART in combination with dasatinib can help UCART cells overcome host T cell mediated immune clearance, thereby providing a prerequisite for UCART to perform tumor killing functions.

FIG. 30E reveals that the absolute number of CAR positive cells in UCART cells was consistently elevated in dasatinib-treated Km-CD5-UCART groups, whereas it was consistently and consistently low in Ctrl-CD5-UCART and DMSO-treated groups. The main functional population in the Km-CD5-UCART group under dasatinib treatment is a CAR positive cell, and the combination of Km editing and dasatinib enables the CAR positive cell to obtain survival advantage and generate enrichment. The CAR positive rate was significantly increased only in Km-CD5-UCART in dasatinib-treated group, and no significant increase in CAR positive rate was seen in both Ctrl-CD5-UCART and DMSO-treated groups. At the eighth day of the test, all groups of CAR positive cells began to decline, suggesting a functional exhaustion of CART cells.

The inventors believe that in this test, the real time target ratio is favorable to reflect the dynamic relationship between the effector components in the system and the mock host T cells, as shown in fig. 30F, the Km-CD5-UCART group under dasatinib treatment exhibited a sustained increase in the effective target ratio, whereas the other control groups did not. Providing a high-fold allogeneic T cell environment (even if initiated with a 1:50-1:100 effective target ratio), km-CD5-UCART cells can continuously reduce the effective target ratio under the combination of dasatinib. The acquisition of this data gives the inventors great confidence in the effective survival and proliferation of Km-UCART in vivo.

In addition, the inventors amplified the LCK-T316 region of the D11 remaining cells in the entire reaction by further analyzing the genomic sequences of each group of cells in the D11 day mixed lymphocyte reaction, and analyzed the LCK-T316I mutation enrichment level of the remaining cells by Sanger sequencing. The results are shown in FIG. 31A, in which CD5-UCART cells without LCK-T316I editing did not show any enrichment of T316I mutation on day D11 of the MLR reaction (six rows in blue in FIG. 31A), whether or not dasatinib drug treatment was not performed. Whereas, the CD5-UCART cells with LCK-T316I mutation do not have enrichment of T316I mutation when MLR reaction is treated with DMSO; whereas when treated with 25nM dasatinib, more than 23% of mutations occurred in T316I; when the concentration was further increased to 50nM, the ratio of mutation enrichment was increased to 43% or more (six rows below FIG. 31A), and the statistical data is shown in FIG. 31B. These data demonstrate that dasatinib is able to inhibit the activation of allogeneic T (host T) cells while Km-edited UCAR-T cells are resistant to the inhibition of dasatinib.

In summary, the inventors have demonstrated that in order to protect UCART from host T cell mediated immune rejection, a regimen of Km editing followed by combined action with dasatinib is feasible.

Example 16: panatinib (PN) at a concentration of 200nM is effective in inhibiting T cell activation in vitro

The foregoing embodiments basically illustrate the strategy and principles of the invention, with two main core inventions: (1) drug specific treatment: the inventor introduces the tyrosine kinase inhibitor dasatinib into the pretreatment of the universal CAR-T, and the dasatinib inhibits the activation of host T and NK cells, so that the universal CAR-T can survive in the environment of the host cells; (2) LCK-T316I mutation introduction: the inventors introduced LCK-T316I (Km) mutations on universal CAR-T cells through a single base editing technique A3A-CBE3 system, making them resistant to inhibition of CAR signaling by dasatinib. Finally, km edited UCAR-T can be normally activated to target killer tumor cells under dasatinib treatment, and host T and NK cells are prevented from being cleared.

The Tyrosine Kinase Inhibitor (TKI) adopted in the invention is not only dasatinib. The inventors found that panatinib could also be a special treatment drug in this invention that implements a generic CAR-T strategy.

In the panatinib study, the inventors first obtained CD3 positive T cells from donor PBMC using the maytansinoid CD3 positive magnetic bead sorting kit, and identified the purity and efficiency of CD3 positive cells obtained by sorting by flow cytometry. The CD3 positive T cells obtained by sorting were then divided into three groups, the control group for electrotransport CBE3 protein (CBE 3), the synonymous mutant control group for electrotransport CBE3 protein and sgRNA12 complex (Sg 12) and the LCK-T316I mutant group for electrotransport CBE3 and sgRNA16 complex (Sg 16-Km), respectively. The inventors used a Lonza electrotrans-analyzer to edit LCK gene with single base, the editing efficiency was as shown in fig. 32A, LCK-T316I mutation efficiency was 30% for Sg16-Km group, and two other groups of control cells did not carry this mutation (fig. 32A).

Experimental procedure As shown in FIG. 32B, the inventors equally divide three edited cells into 6 groups and culture them in flat bottom 96-well plates with two technical replicates per group and 0.15X10 s per well ⁶ And (3) cells. The edited cells of each group were first pretreated with panatinib for 2 hours, with corresponding concentrations of PN of 0, 10nM, 50nM, 100nM and 200nM, respectively (FIG. 32B). Two hours later, an appropriate amount of anti-CD 3/CD38 DynaBeads was used to activate each group of cells while a group of 0nM panatinib-treated blank without bead activation was maintained. Culture was continued for 18 hours later. Activated T cell surface marker molecule 4-1BB was detected with an APC-labeled murine IgG monoclonal antibody, activated T cell surface marker molecule CD25 was detected with a PE-labeled murine IgG monoclonal antibody, and activated T cell surface marker molecule CD69 was detected with a FITC-labeled murine IgG monoclonal antibody.

The results of the flow analysis showed that panatinib treatment at 100nM and below was insufficient to inhibit TCR signaling activation in T cells of the normal control group, while 4-1BB and CD69 were still expressed by induction, indicating that panatinib at 100nM and below was unable to inhibit T cell activation. However, at 200nM panatinib treatment, the CBE3 and Sg12 control groups had 1.00% and 1.85% of cells expressing CD25 and 4-1BB biscationic molecules, respectively, while the Km group had 24.1% of biscationic cells (FIG. 32C); meanwhile, CBE3 and Sg12 control groups had 5.59% and 5.31% of cells expressing CD25 and CD69 double positive molecules, respectively, while Km group had 17.7% of double positive cells (fig. 32D). Taken together, the inventors demonstrated the function of panatinib in inhibiting T cell activation. Meanwhile, the inventors demonstrated that Km-edited T cells (carrying LCK-T316I mutations) can tolerate 200nM inhibition of T cell activation by panatinib. Thus, by selecting a suitable concentration (> 200 nM) of panatinib for pretreatment, inhibition of normal TCR signal activation (inhibition of host T cell function) can be achieved, while LCK-T316I mutated UCAR-T cells are able to tolerate inhibition of their activation by panatinib at that concentration.

To further determine the range of concentrations in which panatinib functions to inhibit T cell activation in vitro and is tolerated by T316I mutations, the inventors tested inhibition of T cell activation at higher concentrations of panatinib.

The inventors used two groups of T cells (CBE 3 control and Km edited) in the above experiments for the study. The experimental procedure was substantially identical to that described above (fig. 32B). Two groups of edited cells were equally divided into 7 groups and cultured in flat bottom 96-well plates, each group containing two technical replicates, each well containing 0.15X10 s ⁶ And (3) cells. The cells after editing were first pretreated with panatinib for 2 hours, and the corresponding concentrations of PN for each group were 0, 10nM, 50nM, 200nM, 500nM and 1000nM, respectively (FIGS. 33A, B). Two hours later, an appropriate amount of anti-CD 3/CD38 DynaBeads was used to activate each group of cells while a group of 0nM panatinib-treated blank without bead activation was maintained. Culture was continued for 18 hours later. Activated T cell surface marker molecule 4-1BB was detected with an APC-labeled murine IgG monoclonal antibody, activated T cell surface marker molecule CD25 was detected with a PE-labeled murine IgG monoclonal antibody, and activated T cell surface marker molecule CD69 was detected with a FITC-labeled murine IgG monoclonal antibody.

The results of the flow assay showed that treatment with panatinib at 50nM and below was insufficient to inhibit TCR signaling activation in T cells of the normal control group, while 4-1BB and CD69 were still expressed by induction, indicating that panatinib at 50nM and below was unable to inhibit T cell activation. However, consistent with the above experiments, at 200nM panatinib treatment, the CBE3 control group showed few cells double positive for CD25/4-1BB, while the Km group still had 19.71% of cells expressing CD25 and 4-1BB molecules (FIG. 33A, fifth column); meanwhile, almost no cells in the CBE3 control group were CD25/CD69 double positive, while 23.9% of cells in the Km group still expressed CD25 and CD69 molecules (FIG. 33B, fifth column), and the results consistent with the previous experiment were completely obtained.

In addition, T cell activation was completely inhibited in the CBE3 control group with 500nM panatinib, and only 2.42% of CD25 and 4-1BB biscationic cells and 6.75% of CD25 and CD69 biscationic cells were also present in the Km-edited group (FIG. 33A, sixth column B). Suggesting that LCK-T316I mutated T cells were significantly less able to be activated under 500nM panatinib treatment, demonstrating that Km editing was not able to effectively tolerate inhibition of TCR signaling by 500nM panatinib. Under 1000nM panatinib treatment, activation of T cells was completely inhibited in both the CBE3 control and Km-edited groups (FIG. 33A, seventh column B), and the cell number was significantly reduced, suggesting that 1000nM panatinib had a greater effect on T cell survival, and LCK-T316I mutation was not effective in tolerating inhibition of TCR signaling by 1000nM panatinib.

Taken together, the inventors believe that 200nM panatinib has been effective in inhibiting T cell activation. For LCK-T316I mutated T cells, the mutation is able to tolerate inhibition of T cell activation by panatinib at a concentration of 200nM, but this tolerability is significantly reduced when the panatinib concentration is increased to 500 nM. Panatinib at 500nM was able to block TCR signaling activation by Km-edited T cells. Therefore, panatinib can be used as a pretreatment scheme of a general CAR-T strategy in the invention, the 200nM concentration of panatinib has the same function as dasatinib, inhibits the host T from killing UCAR-T, ensures that LCK-T316I-UCAR-T cells still have normal functions, and can be activated and mediate the killing of targeted tumor cells. When the panatinib concentration is increased to 500nM, the panatinib can be used as a molecular switch to inhibit the activation of LCK-T316I-UCAR-T cells.

Example 17: panatinib at a concentration of 200nM is effective in inhibiting NK cell activation in vitro

In the above examples the inventors demonstrate that panatinib at a concentration of 200nM is effective in inhibiting T cell activation. To further explore the inhibition of NK cell activation by panatinib and the concentration threshold, it was determined that panatinib was effective in inhibiting activation of both T cells and NK cells and the concentration threshold.

The inventors used CD3 negative cells in human PBMC, comprising more than 5% CD56 positive NK cells. The inventors studied the effect of different concentrations on NK activation by treatment with panatinib at four concentrations of 0, 50nM, 200nM and 500nM, respectively. The flow is as follows: panatinib pretreatment at different concentrations was performed overnight. The next day was resuspended in NK medium and K562 was added for activation, with lymphocyte activator as positive control for activation, also retaining a portion of NK cells not activated by K562, and each group was set up in duplicate. CD107a antibody and monensin were added simultaneously with activation, and CD107a release from CD56 positive NK cells was flow-detected after 6 hours of co-incubation.

As shown in fig. 34A, NK cells with lymphocyte activators added had a CD107a transport of 96% or more (second row in fig. 34A) and a K562 activation of 77% or more (third row in fig. 34) without panatinib treatment. As panatinib concentration increases, NK cells become activated by K562 and the proportion of CD107a expressed gradually decreases. When the panatinib concentration reached 200nM, K562 was substantially unable to activate NK cells causing them to release CD107a (fig. 34A, second row below), demonstrating that the 200nM concentration of panatinib was able to effectively exert inhibition of NK cell activation function in vitro. As shown in fig. 34B, the inventors performed a significance analysis of the above experimental data in a bar graph, basically determining that a concentration of 200nM was able to significantly inhibit NK cell activation compared to the panatinib-free treatment group.

Thus, the above data further demonstrate that panatinib can be used as a pretreatment regimen for the universal CAR-T strategy in the present invention, and that panatinib at a concentration of 200nM can inhibit activation of T and NK cells, avoiding killing of UCAR-T by host T and NK cells. Meanwhile, LCK-T316I mutation can enable UCAR-T cells to resist inhibition of panatinib on T cell activation, and can be activated normally and mediate killing of targeted tumor cells.

Example 18: in vitro inhibition of T cell activation by panatinib has a continuous effect

In example 16, the inventors demonstrated that a 200nM concentration of panatinib was effective in inhibiting T cell activation in vitro, thereby directing the in vivo drug concentration. Considering the metabolism of panatinib in vivo, the inventor designs an in vitro panatinib dynamic concentration experiment to detect whether the inhibition of T cell activation function is still effective under dynamic concentration change.

Experiments were performed in this example using CAR-T cells that had been prepared to be wild-type anti-BCMA. Treatment with panatinib at a concentration of 0nM, 200nM or 500nM, respectively, was carried out overnight, the next day the concentration was reduced. FIG. 35A,0nM pretreatment group did not require further concentration reduction, 200nM pretreatment was followed by overnight adjustment to 0, 50, 100 and 200nM,500nM pretreatment was followed by overnight adjustment to 0, 50, 100, 200nM and 500nM, respectively. Meanwhile, RPMI-8226 cells expressing BCMA antigen are used as positive target cells, and when the concentration is regulated on the next day, the target ratio of the CAR-T cells to the target cells is 1:1 and the control is a blank medium without target cells. CD107a and monensin were added simultaneously. After 4 hours of co-incubation, CD107a release was detected in CD8 and CAR biscationic cells.

As shown in fig. 35, CAR-T cells in the panatinib-free treatment group were activated by RMPI-8266 target cells, and about 40% of cells released CD107a, demonstrating that CAR-T cells could be effectively activated by positive target cells (fig. 35A, first and second rows, upper and lower two parallel wells, respectively).

When the concentration was adjusted to 0nM the next day after pretreatment with 200nM panatinib, the proportion of CD107a positive cells decreased from about 40% to about 16%, demonstrating the continued effect of panatinib on T cell activation (FIG. 35A, third, fourth, leftmost column). When the concentration was adjusted to 50nM or higher the next day after pretreatment with 200nM panatinib, the proportion of CD107a positive cells was decreased to be consistent with the negative control group, suggesting that activation of T cells could already be completely inhibited after the concentration of panatinib was decreased from 200nM to 50nM (fig. 35A, third, fourth, and third).

When the pretreatment was performed with 500nM panatinib, the concentration was adjusted to 0nM the next day, the proportion of CD107a positive cells decreased from about 40% to about 3%, again demonstrating the continued effect of panatinib on T cell activation, and the higher the pretreatment concentration, the more pronounced the continued effect of inhibition (FIG. 35A, fifth, sixth, leftmost column). When the concentration was adjusted to 50nM or higher the next day after pretreatment with 500nM panatinib, the proportion of CD107a positive cells was decreased to be consistent with that of the negative control group, suggesting that activation of T cells could be completely inhibited even after the concentration of panatinib was decreased from 500nM to 50nM (FIG. 35A, fifth, sixth, last three columns).

As shown in fig. 35B, the inventors performed a statistical analysis of the above data, demonstrating that CAR-T cells that were adjusted to a concentration of 0nM after 200nM panatinib pretreatment and were treated for 4 hours had significantly reduced CD107a positive rate released by activation of target cells, and CAR-T cells that were adjusted to a post concentration of 50nM or more were not activated by target cells within 4 hours, as compared to cells of the panatinib-free treatment group. At the same time, 500nM pretreatment substantially completely inhibited activation of CAR-T cells by target cells. The above data indicate that there is a continuing effect of panatinib on the inhibition of T cell activation, and that the higher the drug concentration, the more pronounced this continuing effect.

Example 19: in vitro panatinib has a continuous effect on the inhibition of NK cell activation function

In example 17, the inventors demonstrated that panatinib at a concentration of 200nM was effective to exert inhibition of NK cell activation function in vitro, thereby guiding in vivo drug concentration. In order to fully consider the factor of in-vivo panatinib concentration fluctuation, the inventor designs an in-vitro panatinib dynamic concentration experiment to detect whether the inhibition of NK cell activation function is still effective under dynamic concentration change.

The inventor separates NK cells from human PBMC, and cultures and differentiates the NK cells by using an NK culture medium to obtain the CD56 positive NK cells with the purity higher than 95 percent. Four kinds of pretreatment (1) are respectively carried out without panatinib pretreatment; (2) pretreatment with 50nM panatinib; (3) pretreatment with 100nM panatinib; (4) pretreatment with 200nM panatinib, and incubation overnight. The next day the concentrations of panatinib were reduced and adjusted to 0, 50, 100 and 200nM, respectively. Simultaneously, K562 cells are used as positive target cells to activate NK cells according to the effective target ratio of 1:1, simultaneously adding CD107a and monensin. Two replicates were set for each group. After 5 hours of co-incubation, CD107a release was detected in CD56 positive NK cells.

As shown in fig. 36A, NK cells were significantly activated by K562 after pretreatment with four different concentrations of panatinib and then reduced to 0nM, with release of CD107a gradually decreasing with increasing pretreatment concentration, with the strongest being 58% or more (first, second rows in fig. 36A) and the weakest being 38% or more (seventh, eighth, second rows in fig. 36A). K562 was demonstrated to activate NK, and inhibition of NK activation by panatinib was shown to have a continuing effect, the higher the concentration, the more pronounced the continuing effect. Following pretreatment with four different concentrations of panatinib, the next day concentrations were adjusted to four concentrations of 0, 50, 100 and 200nM, and it was found that the NK cell fraction of each group of pretreated released CD107a activated by K562 gradually decreased with increasing next day concentration. And 200nM panatinib was pre-treated to maintain its concentration to effectively inhibit NK activation. Inhibition of NK by panatinib was demonstrated to still require maintenance at higher concentration levels.

As shown in fig. 36B, the inventors performed a significance analysis of the above experimental data to further elucidate the continuous effect of panatinib on the inhibition of NK cell activation function by K562, which was reduced with increasing pretreatment concentration, after treatment of NK cells at different pretreatment concentrations for 4 hours with subsequent adjustment to uniform concentrations. And panatinib needs to be maintained at 200nM to be able to effectively inhibit NK activation.

Example 20: verifying the possibility of panatinib as a combination drug and switch for general CAR-T treatment in this patent

In example 16, the inventors demonstrated that a 200nM concentration of panatinib was effective in inhibiting T cell activation in vitro and that for LCK-T316I mutated T cells, the mutation was able to tolerate inhibition of T cell activation by panatinib at a concentration of 200nM, thereby guiding the in vivo drug concentration. Panatinib is able to block TCR signaling activation of Km-edited T cells when panatinib concentration is raised to 500 nM. Therefore, can be used as a molecular switch to inhibit the activation of LCK-T316I-UCAR-T cells. In example 18, the inventors demonstrated that there is a continuing effect of panatinib on the inhibition of T cell activation at dynamic concentration changes, and that this continuing effect is more pronounced the higher the drug concentration. But this tolerability is significantly reduced when the panatinib concentration is raised to 500 nM.

In order to verify that LCK-T316I mutations not only enable T cells to tolerate inhibition of TCR activation by panatinib, but also enable CAR-T cells to tolerate inhibition of CD107a transport by panatinib during CAR activation, while fully taking into account factors of panatinib concentration fluctuations in vivo, the inventors devised experiments that demonstrate that LCK-T316I mutated CD19-CAR-T cells can tolerate inhibition of CD107a transport by panatinib at a concentration of 200 nM. However, panatinib was also able to block CD107a transport of LCK-T316I mutated CD19-CAR-T cells when panatinib concentration was increased to 500 nM. Thus, LCKT316I mutation in combination with panatinib can be used as a switch for CD19-UCART treatment.

In this example, the inventors performed experiments using CD19-UCART cells which have been prepared and edited by Km. Both groups of cells were treated overnight with panatinib at 200nM or 500nM concentrations, respectively (fig. 37a,37 b). The following day, 200nM pretreatment overnight groups adjusted to 0, 50, 100 and 200nM,500nM pretreatment overnight after adjusting to 0, 100, 200nM and 300nM, respectively. Meanwhile, raji cells expressing CD19 antigen are used as positive target cells, and the target ratio of the effect of the CD19-UCART cells to the target cells is 1:1 and the control is a blank medium without target cells. Simultaneously, CD107a antibody and monensin were added. Two technical replicates were made for each group. After 4 hours of co-incubation, CD107a release was detected in CD8 and CAR biscationic cells.

As shown in FIG. 37A, when pre-treated with 200nM panatinib overnight, the next day was adjusted to concentrations of 0, 50, 100 and 200nM, and incubated with Raji positive target cells expressing CD19 antigen for 4 hours, the proportion of CD107A positive cells of the CD19-UCART group was reduced from 17.2% (the proportion of CD107A positive cells was taken from the average of the two technical replicates) to 0.735% and the proportion of CD107A positive cells of the Km-edited CD19-UCART group was reduced from 16.6% to 5.12%. At 50nM panatinib, the translocation of CD107a in the CD19-UCART group was substantially inhibited (0.485%), but the proportion of CD107a positive cells in the Km-edited CD19-UCART group was 12.7%. This set of experiments demonstrates that LCK-T316I mutated CD19-UCART cells are resistant to inhibition of CD107a transport during CAR activation by panatinib at a concentration of 200 nM. And at dynamic concentration changes, LCK-T316I mutated CD19-UCART cells have a continuing effect on the ability of panatinib to tolerate the inhibition of CD107a transport during CAR activation.

As shown in FIG. 37B, when pre-treated with 500nM panatinib overnight, the next day was adjusted to concentrations of 0, 100, 200 and 300nM, and after incubation with Raji positive target cells expressing CD19 antigen for 4 hours, the proportion of CD107a positive cells of the CD19-UCART group was reduced from 1.73% to 0.495% and the proportion of CD107a positive cells of the Km-edited CD19-UCART group was reduced from 11.15% to 1.47%. And at dynamic concentration changes in Km edited CD19-UCART group after 500nM panatinib pretreatment overnight LCK-T316I mutated CD19-UCART cells still have a continuing effect on the tolerability of panatinib to inhibit CD107a transport during CAR activation. Although the higher the drug concentration, the lower this tolerability (only 1.47% of CD107a positive cells of the Km-edited CD19-UCART group were treated with panatinib at a concentration of 300 nM). This set of experiments demonstrates a significant reduction in the tolerance of LCK-T316I mutated CD19-UCART cells to inhibition of CD107a transport during CAR activation by panatinib at a concentration of 500 nM.

Taken together, the inventors demonstrate that LCK-T316I mutated CD19-UCART cells are resistant to inhibition of CD107a transport during CAR activation by panatinib at 200nM concentrations. And at dynamic concentration changes, LCK-T316I mutated CD19-UCART cells have a continuing effect on the ability of panatinib to tolerate the inhibition of CD107a transport during CAR activation. However, the panatinib pretreatment at 500nM concentration substantially completely inhibited activation of CD19-UCART cells by target cells, whereas LCK-T316I mutated CD19-UCART cells were not substantially activated by Raji target cells even at 300nM concentration of panatinib pretreatment. Thus, the inventors believe that a 200nM concentration of panatinib and LCK-T316I mutation may be used in combination with activation of CD19-UCART, while a 500nM concentration of panatinib may shut down activation of LCK-T316I mutated CD 19-UCART.

Example 21: LCKT316I mutation and dasatinib combination strategy allows B2M knockout CD19-UCART to be mixed in vitro The most advantageous amplification and persistence in the Heterolymphocyte reaction

Considering that dasatinib has been demonstrated in the above examples to be effective in inhibiting activation of T cells and NK cells, whereas CAR-T cells carrying LCK-T316I mutations are effective against inhibition of activation of CAR molecules by dasatinib, thereby activating and killing tumor cells in normal targeting. Furthermore, the inventors have demonstrated in examples 14 and 15 that the non-knocked-out version B2M of CD5-UCART is effective against allogeneic T cells in an in vitro mixed lymphocyte reaction with them, resulting in an expansion advantage. Therefore, in order to further verify whether the B2M knockout version of UCART can effectively resist NK cells from being cleared on the basis of combined administration with dasatinib to obtain amplification advantages, the inventors have devised the following experiments.

The inventors isolated CD3 positive T cells from PBMC, activated them, knocked out B2M and TRAC genes, and subjected them to lentiviral infection with anti-CD 19 CAR molecules (see SEQ ID Nos. 35-56 for specific sequences). As shown in fig. 38A, the ratio of B2M and TRAC double knockouts was 74% or more (left column in fig. 38A), and the CAR positive rate was 28.8% (right column in fig. 38A). On the sixth day after activation, LCK-T316I was edited for the cells with an editing efficiency of 50% or more.

Subsequently, the inventors performed in vitro mixed lymphocyte reaction tests using these prepared anti-CD 19 UCART cells. The inventors removed CD3 positive T cells from fresh PBMCs of a different donor than UCAR-T, and retained the CD3 depleted PBMCs (mainly containing CD56 positive NK cells and CD19 positive B cells) and UCART for mixed lympho-reactions with four gradients of 20:1, 10:1,5:1 and 2.5:1 effective target ratio (allogeneic cells: UCART). There are two types of CD19 UCART cells tested: LCK edited group (Km) and LCK unedited group were treated with DMSO or 50nM dasatinib, respectively, NK medium, cell counts were performed 24 hours (D1), 48 hours (D2) and 96 hours (D4) after co-incubation, and the proportion of each component was flow-detected, including CD19, CAR molecules, TRAC, CD56, and the like.

As shown in fig. 38B, at 20:1 target ratio, UCART cells of DMSO-treated control group failed to amplify efficiently, suggesting that CD19-UCART was activated by B cells but failed to gain amplification advantage, mainly due to rapid clearance (red and purple lines) of activated NK cells after B2M knockout. Neither did the unedited set of cells (green line) of CD19-UCART treated with dasatinib expand suggesting that dasatinib effectively inhibited activation of CAR molecules. Only CD19-UCART edited by LCK-T316I can be rapidly amplified under the treatment of dasatinib, which suggests that dasatinib cannot inhibit activation of LCK-T316I mutated CD19-UCART, and NK cell activity is inhibited, and B2M knockout UCART cells (blue line) cannot be effectively cleared.

Similarly, as shown in FIG. 38C, the CAR positive rate of CD19-UCART was significantly increased only in dasatinib-treated Km-CD19-UCART, and there was no significant increase in the CAR positive rate in both Km unedited CD19-UCART and DMSO-treated groups.

Furthermore, as shown in FIG. 38D, none of the B cells of the DMSO control group survived, suggesting killing by CD19-UCART (red and purple lines). Under dasatinib treatment, activation of unedited CD19-UCART was inhibited and B cells could not be killed, so that B cells of this group remained at a higher level (green line) at all times. While CD19-UCART cells of the Km editing group can effectively resist the inhibition of dasatinib, kill B cells, and reduce the B cells (blue line).

Finally, as in FIG. 38E, the change in LCK-T316I-CD19-CAR-T cell mass was counted for the LCK mutant group at four different potency target ratios in 50nM dasatinib treatment. The results suggest that there is significant expansion of the total amount of CAR positive cells at each effective target ratio, and basically shows a trend of stronger expansion the lower the effective target ratio. These results show that the combination strategy of LCKT316I mutation and dasatinib can lead to the most expansion and persistence advantage of B2M knockout CD19-UCART in vitro mixed lymphocyte reaction with more NK cells.

^T316I Example 22: LCK mutation and dasatinib combination strategy to MHC Class I molecules (B2M) and MHC-class IICD19-UCART with common knockout of molecule (CIITA) has the advantages of optimal amplification and persistence in-vitro mixed lymphocyte reactionPotential of

The method comprises the following steps:

(1) Resuscitation and activation of T cells;

(2) Performing TRAC and B2M, CIITA gene knockout and T316I editing (Km) of LCK genes on activated T cells;

(3) Lentivirus infects T cells;

(4) TRAC and B2M, CIITA gene knockout efficiency, TRAC and B2M double knockout efficiency about 70%, CIITA gene knockout efficiency 50%. In order to avoid the interference of positive cells on subsequent experiments, cells are subjected to TRAC and B2M, CIITA negative selection, and the result of the cells after negative selection is shown in FIG. 39A, wherein the knockout efficiency of TRAC gene is >97%, the knockout efficiency of B2M gene is >97%, and the knockout efficiency of CIITA gene is >99%.

(5) CAR positive rate detection, results are shown in figure 39A, >15%;

(6) Km editing efficiency was detected, and as a result, as shown in fig. 39B, km editing did not occur in Ctrl group; the mutation efficiency of T316I in CD19-UCART cells in Km group was 20%. At that time, the inventors considered that Km-CD19-UCART cells were obtained that could be used for in vitro testing;

(7) In vitro experiments mimic host in vivo cells: in order to test the function of Km-CD19-UCART in the environment of host T, B, NK cells, the inventors have further required donor-derived T, B, NK cells, which are different from the HLA-ABC protein of the donor involved in the preparation of Km-CD19-UCART, as host T, B, NK cells. The T, B, NK cells of this batch were PBMCs.

(8) The survival of Km-CD19-UCART cells in the host T, NK cell environment was examined using mixed lymphocyte reaction: this mixed lymphocyte reaction was intended to examine the ability of Km-CD19-UCART cells to resist T-cell, NK cell mediated immune rejection in vitro, and thus the inventors considered Km-CD19-UCART as a theoretical target cell. Host T cells, NK cells act as effector cells.

The target cells in this mixed lymphocyte reaction were divided into 2 groups: (1) The Ctrl group is a CD19-UCAT cell control group with synonymous mutation aiming at LCK; (2) Km groups are CD19-UCAT cell experimental groups in which LCKT316I editing occurred. Effector cells in this mixed lymphocyte reaction only use the same donor-derived allogeneic host PBMC cells.

The present embodiment is intended to be 10: 1-effect target ratio development. Group 2Target cells at 1.5X10 ⁵ The number of CAR positive cells was 6 wells each in a flat bottom 48-well plate (500. Mu.l volume), and effector cells were 1.5X10 s each ⁶ The number of individual cells (500. Mu.l volume) was mixed with effector cells and the total volume per well was 1000ul, all cultured in complete medium for T cells. The existing 2 groups of mixed wells of effector cells and target cells were each given 2 treatments, each setting 2 technical replicates: treatment one was DMSO and treatment two was 50nM dasatinib. The well plate was placed in an incubator at 37℃for cultivation. Dasatinib and DMSO in the culture system were supplemented every two days.

This test, the inventors predicted that Km group effector cells could exhibit growth advantages over other groups in dasatinib-treated groups. To verify the above hypothesis, the inventors need to collect the following data during the course of the mixed lymphocyte reaction: cell counts at different time points; ratio and absolute number of effector cells to target cells at different time points; CAR positive rate of effector cells.

days

4, 9, 12, and 16 of co-culture (day of plating is day 0) and counted the cells to determine the proportion of the total cells in each well and calculate the absolute number of cells in each well. The removed cells also need to be used in flow cytometric analysis to determine the ratio of effector cells, target cells, and CAR positive rate of effector cells.

The inventors in this mixed lymphocyte reaction calculated the index of interest by combining the cell count results at each time point with the flow cytometry results. The actual plating cell number on day 0 was obtained by cell counting and flow cytometry after plating, without regard to the amount of cells to be added at the time of experimental design. Because errors in loading can lead to inconsistent actual plating and cell volume for the planned inoculum, and secondly because of the set up of technical replicates, counting the actual inoculum volume makes the results easier to carry out for the biological statistical analysis. The actual CAR positive rate of effector cells was about 18% (Ctrl group), 25% (Km group) when tested. After the end of this test, the inventors performed detailed statistical analysis of the number dynamic change of target cells (fig. 40A), the number dynamic change of effector cells (fig. 40C, 40D), and the CAR positive rate in target cells (fig. 40E), respectively, based on the various indexes obtained at each time point.

As shown in FIGS. 40A and 40B, neither Ctrl-CD19-UCART nor Km-CD19-UCART was amplified efficiently in the environment of host T cells, NK cells in DMSO treatment, whereas control Ctrl-CD19-UCART and Km-CD19-UCART were amplified efficiently in 50nM dasatinib treatment.

For the dynamic change of host T cell NK cells, as shown in FIGS. 40C and 40D, under the DMSO treatment, both the Ctrl-CD19-UCART and Km-CD19-UCART groups of host T cells and NK cells are obviously increased compared with the DS treatment group. Consistent with the phenomenon observed in the previous figures 40A and 40B, it is explained that the reason for the reduced number of UCART in DMSO-treated group is that activation and expansion of host T cells, NK cells, results in the elimination of UCART cells.

FIGS. 40E, 40F reveal that CAR positive cells in UCART cells were consistently elevated in dasatinib treated Km-CD19-UCART groups, while they were slightly elevated in Ctrl-CD19-UCART, and that DMSO treated groups were consistently lowered. The main functional population in the Km-CD19-UCART group under dasatinib treatment is indicated to be CAR positive cells,km editing and dasatinib are used in combination such that Car positive cells gain survival advantage and produce enrichment. The CAR positive rate was significantly increased only in Km-CD19-UCART in dasatinib treated group, and no significant increase in CAR positive rate was seen in Ctrl-CD19-UCART and DMSO treated group.

^T316I Example 23: optimization and confirmation of LCK mutant UCAR-T preparation process

Considering the varying efficiency of LCK-T316I mutation in UCK-T cells prepared in the above examples, the inventors directed to LCK in order to maintain efficient LCK-T316I mutation during application ^T316I The preparation process of the mutated UCAR-T is optimized, and a preparation method capable of ensuring the high mutation rate of LCK-T316I is confirmed.

The final preparation process flow we confirmed is as follows:

day0, sorting T cells using a humane kit using a meria-gentle Pan T Cell Isolation Kit, culturing T cells in a cytokine-free medium, resting for 5 hours, performing resting electrotransformation using a Celetrix electrotransformation apparatus while knocking out the TRAC and B2M genes using Cas9 protein, editing Km genes using CBE3 protein, immediately adding 50nM-100nM dasatinib for treatment after editing, and performing T cell activation using a meria-gentle humane CD2/CD3/CD28 activator for 2 hours, adding 25ul/ml by volume, and stimulating for 2 days;

day1, no operation, observation culture;

day2, 48hr after T cell activation, lentiviral transduction with moi=1-3, with transfer aid 1% dmso or 100x lentiboost;

Day3, virus liquid change is carried out 24hr after virus transformation, and cells are continuously cultured;

day4, no operation, observation and culture;

day6, performing negative selection of TRAC and B2M double-negative cells, selecting TRAC negative cells by using CD3 microblades, selecting B2M negative cells by using PE-anti-B2M antibodies and PE microblades, and culturing the cells after negative selection in Grex;

day7, no operation, observation and culture;

day8, semi-liquid exchange or liquid replacement;

day9, no operation, observation culture;

day10/11, cell preparation and cryopreservation.

The inventors performed knockout of B2M and TRAC genes by isolating CD3 positive T cells from PBMCs derived from three different donors using the PanT kit, while editing the cells for LCK-T316I. Cells were then split into two parts, one with 100nM DS (control) and the other with corresponding amount of DMSO as control (control), and after 2 hours the control and control were added with T cell activator (CD 2/CD3/CD28, 25 ul/1M/mL). After 48 hours of activation, it was subjected to anti-CD 19 CAR molecule lentiviral infection, and 24 hours after virus infection, the liquid was changed while DS or DMSO was removed.

As shown in FIG. 41A, in the experimental group B2M and TRAC double knockouts were more than 50% (left column of FIG. 41A), the control group B2M and TRAC double knockouts were not in proportion One (right column of fig. 41A), but all lower than the experimental group. From the flow chart it can be seen that the reason for the lower proportion of double knockouts of control B2M and TRAC than in the experimental group is that DS inhibited activation of TCR-positive cells (confirming the conclusion of example 3). The CAR positive rate was greater than 15% (fig. 41B). On the eighth day of culture, the cells were tested for efficiency of LCK-T316I. The addition of 100nM of the DS group (blue) LCK-T316I was significantly higher than the addition of the corresponding amount of DMSO group (red) (FIG. 41C).Because of The inventors have thus demonstrated that the addition of 100nM dasatinib during UCART preparation significantly enriches the occurrence of T316I mutations Cells, thereby increasing mutation rate in the final product.

^T316I Example 24: optimization and validation of LCK mutated UCAR-T carried third Signal element

And (3) carrying out repeated antigen stimulation reaction by using a prepared sufficient number of Raji cells which contain different third signal structures and are treated by CD19-UCART and mitomycin-C, so as to simulate the continuous expansion process of UCART cells to kill target cells in vivo under the repeated contact environment of the target cells and the UCART cells.

The invention uses gene editing technology to knock TRAC gene and B2M gene into T cell to prepare CD19-UCART cell.

CD19-UCART cells are prepared from healthy donors in small quantity, and in cell samples prepared in batch, the inventor determines that the knockout efficiency of TRAC genes is more than 90% through a flow cytometry method; the detection of CD19 antigen markers measured that different UCART cells expressed about 10-50% of the CAR.

The present invention contemplates measuring UCAR-T cell proliferation and killing function characteristics of UCAR-T cells incorporating different third signaling elements by repeated stimulation of target cell antigens, thereby preferentially carrying LCK of the third signaling element ^T316I Mutant UCAR-T. Two experiments were performed in total, differing from experiment I and experiment II, respectively.

The method comprises the following steps:

(1) Plasmid and lentiviral constructs carrying a different third signal element, the schematic of the plasmid is shown in FIG. 42A;

(2) Cd3+ T cell sorting;

(3) TRAC, B2M gene knockout, T316I editing (Km) of LCK gene are carried out on the T cells which are not activated;

(4) Activating the cells after electroporation;

(5) Infecting the cells with the constructed different lentiviruses;

(6) The TRAC and CD5 gene knockout efficiency results are shown in FIG. 42B;

(7) CAR positive rate detection, due to the lack of molecular markers, car+ ratio was indicated using the CAR structure of Anti-CD19 expressed by CD19 antigen binding, the results are shown in fig. 42C;

(8) Enrichment of TRAC/B2M double negative cells: the inventors used FITC-B2M antibody+FITC microbeds in combination with CD3+ microbeds to perform a filtration operation in LS column to obtain UCART cells with TRAC/B2M-/-ratio > 99%;

(9) Target cell treatment for antigen stimulation in vitro experiments: the inventors have required to obtain a CD19 antigen capable of binding to CD19-UCART in order to test proliferation and killing of CD19-UCART under repeated antigen stimulation. Raji cells were thus treated with mitomycin C at a concentration of 1 ug/M/ml. Such treatment aims at disabling the ability of Raji cells to divide, but retains their surface antigen properties;

(10) The limit proliferation of CD19-UCART cells after repeated killing of tumor cells in vivo is simulated by repeated antigen stimulation: the repeated antigen stimulation experiment aims at comparing the ultimate proliferation capacity of CD19-UCART cells containing different third signal structures under the condition of repeated stimulation by external antigens and whether the cells can still keep stronger capacity of killing tumor cells after continuous proliferation. Thus, the inventors have always activated CD19-UCART cells at a stimulation frequency of once every 3 days, and considered it as a process of continuously eliminating tumor in vivo by CD 19-UCART.

The flow chart of this example is shown in fig. 43A, and effector cells in both experiments I and II were grouped according to different third signal structures. Raji cells pretreated with mitomycin C (1 ug/M/ml) for 16h at Day-1 were used as antigen for target cells stimulated repeatedly. Day0 pretreated target cells were washed 3 times with 10ml PBS before co-cultivation began. Taking each group of CD19-UCART cells according to the total cell quantity of 2M, and then according to the effective target ratio of 2:1 UCART cells and Raji cells were mixed and co-cultured in 3ml of medium. The procedure described above was repeated every three days thereafter to replenish the target cells and to perform counting and flow assays, four times in total with antigen stimulation throughout the experiment.

This embodiment is described in 2:1 effective target ratio development it is stated here that the inventors define an effective target ratio as the absolute number of CD19-UCART cells expressing the CAR molecule versus the absolute number of mock host T cells. Two wells of each group of effector cells were plated in a flat bottom 12-well plate (1.5 ml volume) with a total cell number of 2M, and the target cells were mixed with effector cells with a cell number (effector cell number car+ ratio/2) of 1.5ml volume, 3ml total volume per well, and an effector cell culture density of 0.67M/ml. The total cell amount of effector cells was fixed at 2M each time the target cells were supplemented repeatedly, and if effector cells were less than 2M after the end of the previous stimulation, all were used for the next plating.

When a patient takes an immunosuppressant, a series of immune cells are suppressed and the ability to secrete cytokines is reduced. Or some sort of stranguria-clearing pretreatment drug may also cause the patient to have reduced macrophages, resulting in poor cytokine secretion, such as etoposide. To compare whether the various UCART cell expansions containing the third signal are different in the cytokine-lean and cytokine-rich environments. The inventors set the co-culture conditions to two groups, M1 and M2, respectively. Wherein M1 is CTS-based T cell culture medium without any cytokine supplementation, and M2 is CTS-based T cell culture medium +200U/ml IL2. In addition, each group of effector cells was also provided with no target cell group under the corresponding medium conditions to observe the basal proliferation.

In this test, the inventors predicted that UCART cells containing the third signal would proliferate more than the control group, but it was necessary to confirm UCART cells having the best proliferation ability. The inventors thus need to collect the following data during repeated antigen stimulation experiments: cell counts at different time points; CAR positive rate of effector cells at different time points, killing function of different effector cells after repeated antigen stimulation.

To obtain the above data, the inventors have counted a fraction of cells from each well on the 3 rd, 6 th, 9 th, 12 th, and 15 th days (day of plating is defined as day 0) of co-culture, and the proportion of the total cells in each well is determined, and the absolute number of cells in each well is calculated from the proportion and the count result of the cells. The removed cells also need to be used in flow cytometric analysis to determine the CAR positive rate of effector cells.

The inventors calculated the index of interest by combining the cell count results at each time point with the flow cytometric analysis results, and figures 43B and C show the expansion of the various groups of cd19car+ cells during repeated stimulation in experiment I. In experiment I, 2759 was more rapidly enriched in both M1 and M2 medium relative to the CAR+ enrichment of other CD19-UCART cells. 2661 also maintains a higher level of car+ rate after killing the target cells. However, 2758 group showed no significant increase in car+ under M1 and M2 culture conditions. In each of the groups of UCART cells containing the third signal in experiments II in figures 43D and E car+ cells expanded more than the control group in both M1 and M2 medium, with 2661UCART cells significantly expanding best and the fold of car+ enrichment also being high and still maintaining a strong expansion profile after multiple rounds of stimulation. 2759 still had the amplification advantage of car+ cells in M1 medium, but the amplification advantage was not apparent in M2 medium.

The foregoing experiments have demonstrated that 2759 and 2661 have the amplification advantage of car+ after repeated antigen stimulation, and to further verify whether 2759 and 2661 have the killing function advantage of cells after antigen stimulation, the inventors have targeted the effector cells and Raji-luc cells after four rounds of repeated stimulation of experiment I at an effective target ratio of 1:1 co-culturing in a T cell complete culture medium, and performing four rounds of repeated stimulation on effector cells and Raji-luc cells in an effective target ratio of 2:1 and 0.5:1 after co-culturing in a T cell complete culture medium, detecting luminosity of luciferases at 24h and 48h, so as to judge killing functions of different effector cells on Raji cells. The inventors converted the luminosity of each luciferases to the corresponding killer cell ratio and demonstrated the killing function of the different effector cells in figures 44A-C. In experiment I, 1175 had significantly weaker killing function than UCART cells containing the third signal structure under M1 medium conditions when measured at 24h, and 2759 had the strongest relative killing function at 2661 times under both medium conditions. The cell killing ratio of various UCART cells is not quite different when 48 hours are measured. In experiment II, 2759 has obvious killing function advantage compared with other two UCART cells under the condition of M1 culture, and the killing functions of the three UCART cells are not greatly different under the condition of M2 culture medium.

To further verify whether 2759 and 2661 cells have the advantage of killing function after repeated antigen stimulation, the inventors co-incubated UCART cells after four rounds of repeated antigen stimulation with different target cells (positive target cells Raji expressing CD19 antigen and negative target cells CCRF not expressing CD19 antigen) and different sets of CD19-CAR-T cells in experiment II, stimulated activated CAR signals, mediated CAR-T killing of target cells and CAR-T accumulation of CD107a. The addition of monensin (1:1000) and CD107a-PEcy7 antibodies (1:100) 5 hours prior to detection followed by staining for CD8 and CAR positivity was used to detect enrichment of CD107a transport of CD8 positive CAR positive cells, and shown in FIG. 44D after counting. The release rate of each UCART cell 107a was not greatly different, and the release rate of 2661 was relatively highest under the M1 culture condition.

The foregoing experiments have demonstrated that 2759 and 2661 have the amplification advantage and killing function advantage of car+ after repeated antigen stimulation, and in order to further verify whether 2759 and 2661 have cytokine secretion advantage in cells after antigen stimulation, the inventors collected and rapidly stored cell culture supernatants after four rounds of repeated antigen stimulation for the foregoing experiment I and experiment II in a-80 ℃ refrigerator, and freeze-thawed for cytokine detection within one week. TNF-alpha was detected in experiment I, FIG. 45A; IL2, TNF-alpha, IFN-gamma were detected in experiment II, see FIGS. 45B-D. Only TNF-alpha and IFN-gamma were detected because the medium contained IL2 under the M2 culture conditions.

The inventors used the human IL2 kit, human TNF-alpha kit, human IFN-gamma kit from Cisbio to detect cytokine supernatants after freeze thawing. Because IFN-gamma is generally present in too high a supernatant concentration, the inventors performed IFN-gamma detection after 20-fold dilution of cytokine supernatant. The detection process operates entirely according to the specification flow.

TNF-alpha and IFN-gamma of 2759 were found to have obvious secretory advantages, 2661 times, in the M1 and M2 culture environments. 2661

Neither

2759, 2842 secreted IL2 beyond the control group (1175). 2758 was lower in IL2TNF-alphaIFN-gamma secretion than the control.

In view of the above experimental results, the inventors have demonstrated that part of the third signal structure is indeed capable of promoting the expansion capacity of UCART cells, and that a considerable killing function is retained after a large number of amplifications. The inventors finally selected 2661 and 2759 as further improvements to LCK ^T316I The mutated UCAR-T requires a third signal element to carry.

Some of the amino acid or nucleotide sequences mentioned herein are as follows:

SEQ ID NO:1LCK-sgRNA12-21nt target DNA sequence

TCTACATCATCACTGAATACA

SEQ ID NO:2LCK-sgRNA12-20nt target DNA sequence

CTACATCATCACTGAATACA

SEQ ID NO:3LCK-sgRNA12-19nt target DNA sequence

TACATCATCACTGAATACA

SEQ ID NO:4LCK-sgRNA12-18nt target DNA sequence

ACATCATCACTGAATACA

SEQ ID NO:5LCK-sgRNA16-21nt target DNA sequence

CATCACTGAATACATGGAGAA

SEQ ID NO:6LCK-sgRNA16-20nt target DNA sequence

ATCACTGAATACATGGAGAA

SEQ ID NO:7LCK-sgRNA16-19nt target DNA sequence

TCACTGAATACATGGAGAA

SEQ ID NO:8LCK-sgRNA16-18nt target DNA sequence

CACTGAATACATGGAGAA

SEQ ID NO:9LCK-T316I mutein sequences

MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGSNPP

ASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTGQEGFIPFNF

VAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQN

QGEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQK

PWWEDEWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFL

AEANLMKQLQHQRLVRLYAVVTQEPIYIIIEYMENGSLVDFLKTPSGIKLTINKLLDMA

AQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREGAKFPI

KWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFFTATEGQYQPQP*

Here represents protein termination

SEQ ID NO:10LCK-T316A mutein sequences

MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGSNPP

ASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTGQEGFIPFNF

VAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQN

QGEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQK

PWWEDEWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFL

AEANLMKQLQHQRLVRLYAVVTQEPIYIIAEYMENGSLVDFLKTPSGIKLTINKLLDM

AAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREGAKFP

IKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFFTATEGQYQPQP*

Here represents protein termination

SEQ ID NO:11LCK-T316M mutein sequences

MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGSNPP

ASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTGQEGFIPFNF

VAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQN

QGEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQK

PWWEDEWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFL

AEANLMKQLQHQRLVRLYAVVTQEPIYIIMEYMENGSLVDFLKTPSGIKLTINKLLDM

AAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREGAKFP

Here represents protein termination

SEQ ID NO:12LCK-sacAS9-sgRNA1 target DNA sequence

CTACATCATCACTGAATACATG

SEQ ID NO:13LCK-sacAS9-sgRNA2 target DNA sequence

ATCACTGAATACATGGAGAATG

SEQ ID NO:14LCK-sgRNA1 target DNA sequence

GCTACCCGAGTCGGCTACCA

SEQ ID NO:15LCK-sgRNA11 target DNA sequence

GCTCTACGCTGTGGTCACCC

SEQ ID NO:16LCK-sgRNA8 target DNA sequence

GTATTCAGTGATGATGTAGA

SEQ ID NO:17LCK-sgRNA9 target DNA sequence

TATTCAGTGATGATGTAGAT

SEQ ID NO:18LCK-sgRNA12 target DNA sequence

CTACATCATCACTGAATACA

SEQ ID NO: DNA sequence A C G CCTTCCTGGCCGAGGCCAACCTCATGAAGCAGCTGCAACACCAGCGGCTG GTTCGGCTCTACGCTGTGGTCACCCAGGAGCCCATCTACATCATCattGAATACATGG AGAATGgtgggtgctacccga G T C of 19ssDNA-T316I-for-sgRNA8 as repair template

Here, represents the thio modification between bases, three at each of the 5 'and 3' ends

SEQ ID NO: DNA sequence G A C GCCTTCCTGGCCGAGGCCAACCTCATGAAGCAGCTGCAACACCAGCGGCT GGTTCGGCTCTACGCTGTGGTCACCCAGGAGCCCATCTACATCATCattGAATACATG GAGAATGgtgggtgctacccg a G T of 20ssDNA-T316I-for-sgRNA9 as repair template

SEQ ID NO: the DNA sequence a aggtgtatttccaccctctggcagggacagcagggagagcagtatcccctggtagccgactcgggtagcacccacCATT CTCCATGTATTCaatGATGATGTAGATGGGCTCCTGGGTGACCACAGCGT a G a of 21ssDNA-T316I-for-sgRNA12 as repair template here represents the inter-base thio modification, three at each of the 5 'and 3' ends

SEQ ID NO: DNA sequence A C G CCTTCCTGGCCGAGGCCAACCTCATGAAGCAGCTGCAACACCAGCGGCTG GTTCGGCTCTACGCTGTGGTCACCCAGGAGCCCATCTACATCATCatgGAATACATGG AGAATGgtgggtgctacccga G T C of 22ssDNA-T316M-for-sgRNA8 as repair template

SEQ ID NO: DNA sequence G A C GCCTTCCTGGCCGAGGCCAACCTCATGAAGCAGCTGCAACACCAGCGGCT GGTTCGGCTCTACGCTGTGGTCACCCAGGAGCCCATCTACATCATCatgGAATACATG GAGAATGgtgggtgctacccg a G T of 23ssDNA-T316M-for-sgRNA9 as repair template

SEQ ID NO: the DNA sequence a aggtgtatttccaccctctggcagggacagcagggagagcagtatcccctggtagccgactcgggtagcacccacCATT CTCCATGTATTCcatGATGATGTAGATGGGCTCCTGGGTGACCACAGCGT a G a of 24ssDNA-T316M-for-sgRNA12 as repair template here represents the inter-base thio modification, three at each of the 5 'and 3' ends

SEQ ID NO: DNA sequence A.cndot.G. CCTTCCTGGCCGAGGCCAACCTCATGAAGCAGCTGCAACACCAGCGGCTG GTTCGGCTCTACGCTGTGGTCACCCAGGAGCCCATCTACATCATCgccGAATACATG GAGAATGgtgggtgctacccga.cndot.t.c of 25ssDNA-T316A-for-sgRNA8 as repair template

SEQ ID NO:26ssDNA-T316A-for-sgRNA9 as repair template DNA sequence gac GCCTTCCTGGCCGAGGCCAACCTCATGAAGCAGCTGCAACACCAGCGGCT GGTTCGGCTCTACGCTGTGGTCACCCAGGAGCCCATCTACATCATCgccGAATACAT GGAGAATGgtgggtgctacccg ajt

SEQ ID NO: the DNA sequence a aggtgtatttccaccctctggcagggacagcagggagagcagtatcccctggtagccgactcgggtagcacccacCATT CTCCATGTATTCcggGATGATGTAGATGGGCTCCTGGGTGACCACAGCGT a G a of 27ssDNA-T316A-for-sgRNA12 as repair template here represents the inter-base thio modification, three at each of the 5 'and 3' ends

SEQ ID NO:28ssDNA-T316I-for-sgrna1+sgrna11 as repair template DNA sequence aggaaagctcaagaaaccctccttgcttgagctttgggccagaagaaaaggtgtatttccaccctctggcagggacagcagggagagcagtatcccctggtagccgactcgggtagcacccacCATTCTCCATGTATTCaatGATGATGTAGATGGGCTCCTGGGTGACCACAGCGTAGAGCCGAACCAGCCGCTGGTGTTGCAGCTGCTTCATGAGGTTGGCCTCGGCCAGGAAGGCGTCCGGGGACATGCTGCCCTGCTTCAGSEQ ID NO:29ssDNA-T316M-for-sgrna1+sgrna11 as repair template DNA sequence aggaaagctcaagaaaccctccttgcttgagctttgggccagaagaaaaggtgtatttccaccctctggcagggacagcagggagagcagtatcccctggtagccgactcgggtagcacccacCATTCTCCATGTATTCcatGATGATGTAGATGGGCTCCTGGGTGACCACAGCGTAGAGCCGAACCAGCCGCTGGTGTTGCAGCTGCTTCATGAGGTTGGCCTCGGCCAGGAAGGCGTCCGGGGACATGCTGCCCTGCTTCAGSEQ ID NO:30ssDNA-T316A-for-sgrna1+sgrna11 as repair template DNA sequence aggaaagctcaagaaaccctccttgcttgagctttgggccagaagaaaaggtgtatttccaccctctggcagggacagcagggagagcagtatcccctggtagccgactcgggtagcacccacCATTCTCCATGTATTCcggGATGATGTAGATGGGCTCCTGGGTGACCACAGCGTAGAGCCGAACCAGCCGCTGGTGTTGCAGCTGCTTCATGAGGTTGGCCTCGGCCAGGAAGGCGTCCGGGGACATGCTGCCCTGCTTCAGSEQ ID NO: DNA sequence aggaaagctcaagaaaccctccttgcttgagctttgggccagaagaaaaggtgtatttccaccctctggcagggacagcagggagagcagtatcccctggtagccgactcgggtagcacccacCATTCTCCATGTATTCaatGATGATGTAGATGGGCTCCTGGGTGACCACAGCGTAGAGCCGAACCAGCCGCTGGTGTTGCAGCTGCTTCATGAGGTTGGCCTCGGCCAGGAASEQ ID NO of 31ssDNA-T316I-for-sgRNA1+sgRNA12 as repair template: DNA sequence aggaaagctcaagaaaccctccttgcttgagctttgggccagaagaaaaggtgtatttccaccctctggcagggacagcagggagagcagtatcccctggtagccgactcgggtagcacccacCATTCTCCATGTATTCcatGATGATGTAGATGGGCTCCTGGGTGACCACAGCGTAGAGCCGAACCAGCCGCTGGTGTTGCAGCTGCTTCATGAGGTTGGCCTCGGCCAGGAA with 32ssDNA-T316M-for-sgRNA1+sgRNA12 as repair template

SEQ ID NO: DNA sequence of 33ssDNA-T316A-for-sgRNA1+sgRNA12 as repair template

aggaaagctcaagaaaccctccttgcttgagctttgggccagaagaaaaggtgtatttccaccctctggcagggacagcagggagag

cagtatcccctggtagccgactcgggtagcacccacCATTCTCCATGTATTCcggGATGATGTAGATGGGC

TCCTGGGTGACCACAGCGTAGAGCCGAACCAGCCGCTGGTGTTGCAGCTGCTTCA

TGAGGTTGGCCTCGGCCAGGAA

SEQ ID NO:34Ecoli-A3A-CBE3-protein E.coli purified protein sequence

MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHN

QAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFL

QENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQG

CPFQPWDGLDEHSQALSGRLRAILQNQGNSGSETPGTSESATPESDKKYSIGLAIGTNS

VGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT

RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQT

YNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV

NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG

ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRR

QEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK

GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL

KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYT

GWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG

QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK

GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD

INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ

LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY

DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIR

KRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNS

DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF

EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY

VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV

LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKVHHHHHHHHHH*

Here represents protein termination

SEQ ID NO:35 CD19-78 VL nucleic acid sequence

CAGGCTGTGCTGACTCAGCCACCCTCGGTGTCTGAAGCCCCCAGGCAGAGGGTCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGCTGTAAGCTGGTACCAGCAGCTCCCAGGAAAGGCTCCCAAACTCCTCATCTATTATGATGATCTGCTCCCCTCAGGGGTCTCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTAGGT

SEQ ID NO:36 CD19-78 VL protein sequence

QAVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVSWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKVTVLG

SEQ ID NO:37 CD19-78 VH nucleic acid sequence

GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGCGCCTGTCTTACTCTTGGTCTTCTTGGTACTGGGATTTCTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:38 CD19-78 VH protein sequence

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSYSWSSWYWDFWGQGTLVTVSS

SEQ ID NO:39 CD19-Linker1 nucleic acid sequences

GGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCC

SEQ ID NO:40 CD19-Linker1 protein sequence

GGGGSGGGGSGGGGS

SEQ ID NO:41 CD19-CD8a range nucleic acid sequence

ACTACTACCCCTGCACCTAGGCCTCCCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGAC

SEQ ID NO:42 CD19-CD8a range protein sequence

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

SEQ ID NO:43 CD19-CD8a TM nucleic acid sequence

ATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGC

SEQ ID NO:44 CD19-CD8a TM protein sequence

IYIWAPLAGTCGVLLLSLVITLYC

SEQ ID NO:45 CD19-CD28 intracellular domain (IC) nucleic acid sequences

AGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCAAGACGGCCTGGCCCCACCCGGAAGCACTACCAGCCTTACGCCCCTCCCAGAGACTTCGCCGCCTACCGGTCC

SEQ ID NO:46 CD19-CD28 intracellular domain (IC) protein sequences

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

SEQ ID NO:47 CD19-CD3z intracellular signaling domain nucleic acid sequences

AGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCCGAGATGGGCGGAAAGCCCAGACGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGCGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGA

SEQ ID NO:48 CD19-CD3z intracellular signaling domain protein sequence

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:49 CD19-T2A nucleic acid sequences

GAGGGAAGGGGCAGCTTATTAACATGTGGCGATGTGGAAGAGAACCCCGGTCCC

SEQ ID NO:50 CD19-T2A protein sequence

EGRGSLLTCGDVEENPGP

SEQ ID NO:51 CD19-CSF2RA signal nucleic acid sequence

ATGCTGCTGCTCGTGACCTCTTTACTGTTATGTGAGCTGCCCCACCCCGCTTTTTTACTGATCCCT

SEQ ID NO:52 CD19-CSF2RA signal protein sequence

MLLLVTSLLLCELPHPAFLLIP

SEQ ID NO:53 CD19-tEGFR nucleic acid sequence

CGTAAGGTGTGTAACGGAATCGGCATTGGCGAGTTCAAGGACTCTTTAAGCATCAACGCCACAAACATCAAGCACTTCAAGAATTGTACCTCCATCAGCGGCGATTTACACATTCTCCCCGTGGCTTTTCGTGGCGATTCCTTCACCCACACCCCCCCTCTGGACCCCCAAGAGCTGGACATTTTAAAAACCGTGAAGGAGATCACCGGCTTTCTGCTGATCCAAGCTTGGCCCGAGAATCGTACCGACCTCCACGCCTTCGAGAATTTAGAGATTATTCGTGGAAGGACCAAGCAGCACGGCCAGTTCTCTTTAGCCGTCGTGTCTTTAAACATTACCAGCCTCGGTTTAAGGTCTTTAAAGGAGATTAGCGACGGCGACGTGATCATCTCCGGCAACAAGAACCTCTGCTACGCCAACACCATCAACTGGAAGAAGCTGTTCGGAACCAGCGGCCAAAAGACCAAGATCATCAGCAATCGTGGAGAGAACTCTTGTAAGGCCACTGGTCAAGTTTGCCACGCCCTCTGTAGCCCCGAAGGATGTTGGGGCCCCGAGCCTAGGGACTGTGTTAGCTGCAGAAACGTGAGCAGAGGCAGAGAGTGTGTGGACAAATGCAATTTACTGGAAGGAGAGCCTAGGGAGTTCGTGGAGAACAGCGAATGTATCCAGTGCCACCCCGAGTGTTTACCTCAAGCCATGAACATCACTTGTACCGGAAGGGGCCCCGATAACTGCATCCAATGCGCCCACTACATCGACGGACCCCACTGCGTGAAAACTTGTCCCGCCGGAGTGATGGGAGAGAATAACACTTTAGTGTGGAAGTACG

CCGACGCTGGCCACGTCTGCCATCTGTGCCACCCCAACTGTACCTACGGCTGCACT

GGTCCCGGTTTAGAGGGATGTCCTACCAACGGCCCCAAGATCCCCTCCATCGCCAC

CGGAATGGTGGGCGCTCTGTTATTACTGCTGGTGGTGGCTCTGGGCATCGGTTTATT

CATG

SEQ ID NO:54CD19-tEGFR protein sequence

RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDI

LKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLK

EISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPE

GCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNIT

CTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCT

YGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

SEQ ID NO:55CD19-CD28z-CAR nucleic acid sequence

ATGGCCCTGCCTGTGACAGCTCTGCTCCTCCCTCTGGCCCTGCTGCTCCATGCCGC

CAGACCCCAGGCTGTGCTGACTCAGCCACCCTCGGTGTCTGAAGCCCCCAGGCAG

AGGGTCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGCTGTAA

GCTGGTACCAGCAGCTCCCAGGAAAGGCTCCCAAACTCCTCATCTATTATGATGA

TCTGCTCCCCTCAGGGGTCTCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAG

CCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGC

AGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGGACCAAGGTCAC

CGTCCTAGGTGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCC

GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTG

AAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGG

TGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTG

ACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGA

CAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACC

GCCATGTATTACTGTGCGCGCCTGTCTTACTCTTGGTCTTCTTGGTACTGGGATTT

CTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCATTCGTGCCCGTGTTCCTGCCCG

CCAAACCTACTACTACCCCTGCACCTAGGCCTCCCACCCCAGCCCCAACAATCGC

CAGCCAGCCTCTGTCTCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCC

GTGCACACCAGAGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCTCTGG

CCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA

CCGGAACAGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGAC

CCCAAGACGGCCTGGCCCCACCCGGAAGCACTACCAGCCTTACGCCCCTCCCAGA

GACTTCGCCGCCTACCGGTCCAGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTG

CCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGG

AAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCCGAGATGGGCGGA

AAGCCCAGACGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGAC

AAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGCGG

CAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTA

CGACGCCCTGCACATGCAGGCCCTGCCCCCCAGAGGATCCGGAGAGGGAAGGGG

CAGCTTATTAACATGTGGCGATGTGGAAGAGAACCCCGGTCCCATGCTGCTGCTC

GTGACCTCTTTACTGTTATGTGAGCTGCCCCACCCCGCTTTTTTACTGATCCCTCGT

AAGGTGTGTAACGGAATCGGCATTGGCGAGTTCAAGGACTCTTTAAGCATCAACG

CCACAAACATCAAGCACTTCAAGAATTGTACCTCCATCAGCGGCGATTTACACAT

TCTCCCCGTGGCTTTTCGTGGCGATTCCTTCACCCACACCCCCCCTCTGGACCCCC

AAGAGCTGGACATTTTAAAAACCGTGAAGGAGATCACCGGCTTTCTGCTGATCCA

AGCTTGGCCCGAGAATCGTACCGACCTCCACGCCTTCGAGAATTTAGAGATTATT

CGTGGAAGGACCAAGCAGCACGGCCAGTTCTCTTTAGCCGTCGTGTCTTTAAACA

TTACCAGCCTCGGTTTAAGGTCTTTAAAGGAGATTAGCGACGGCGACGTGATCAT

CTCCGGCAACAAGAACCTCTGCTACGCCAACACCATCAACTGGAAGAAGCTGTTC

GGAACCAGCGGCCAAAAGACCAAGATCATCAGCAATCGTGGAGAGAACTCTTGT

AAGGCCACTGGTCAAGTTTGCCACGCCCTCTGTAGCCCCGAAGGATGTTGGGGCC

CCGAGCCTAGGGACTGTGTTAGCTGCAGAAACGTGAGCAGAGGCAGAGAGTGTG

TGGACAAATGCAATTTACTGGAAGGAGAGCCTAGGGAGTTCGTGGAGAACAGCG

AATGTATCCAGTGCCACCCCGAGTGTTTACCTCAAGCCATGAACATCACTTGTAC

CGGAAGGGGCCCCGATAACTGCATCCAATGCGCCCACTACATCGACGGACCCCA

CTGCGTGAAAACTTGTCCCGCCGGAGTGATGGGAGAGAATAACACTTTAGTGTGG

AAGTACGCCGACGCTGGCCACGTCTGCCATCTGTGCCACCCCAACTGTACCTACG

GCTGCACTGGTCCCGGTTTAGAGGGATGTCCTACCAACGGCCCCAAGATCCCCTC

CATCGCCACCGGAATGGTGGGCGCTCTGTTATTACTGCTGGTGGTGGCTCTGGGC

ATCGGTTTATTCATGTGASEQ ID NO:56CD19-CD28z-CAR protein sequence

MALPVTALLLPLALLLHAARPQAVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVSWYQ

QLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSL

NGWVFGGGTKVTVLGGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSG

YSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWS

SLKASDTAMYYCARLSYSWSSWYWDFWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPT

PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN

HRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY

QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA

EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCG

DVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCT

SISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENL

EIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGT

SGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCN

LLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAG

VMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM*

Here represents protein termination

SEQ ID NO:57CD5-Ab20001-61-VH nucleic acid sequence

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTG

AAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAACTATGCTATCAGCTGGGT

GCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCCTACAAT

GGTGACACAAAATATGCACAGAGGCTCCAGGGCAGAGTCACCATGACCACAGAC

ACATCCACGAGCACAGCCTACATGGAGCTGAGGAACCTAAGATCTGACGACACGG

CCGTGTATTACTGTGCGCGCTACGAATCTATGTCTGGTCAGGATATCTGGGGTCAAG

GTACTCTGGTGACCGTCTCCTCASEQ ID NO:58CD5-Ab20001-61-VH protein sequence

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGWISAYNG

DTKYAQRLQGRVTMTTDTSTSTAYMELRNLRSDDTAVYYCARYESMSGQDIWGQGT

LVTVSSSEQ ID NO:59CD5-Ab20001-42-VH nucleic acid sequence

GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGGTAGCCTGC

GTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTAGCCATAGCGCCATGGGTTGGGTT

CGTCAGGCACCTGGTAAAGGTCTGGAATGGGTTAGCAGCATCTATGCCCGCGGCG

GCTATACCTATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAACA

GCAAAAATACCCTGTACCTGCAGATGAATAGTCTGCGTGCAGAGGATACCGCAGTG

TATTATTGTGCGCGCGGTTACCATCTGGAATACATGGTTTCTCAGGATGTTTGGGGT

CAAGGTACTCTGGTGACCGTCTCCTCASEQ ID NO:60CD5-Ab20001-42-VH protein sequence

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHSAMGWVRQAPGKGLEWVSSIYARGG

YTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYHLEYMVSQDVW

GQGTLVTVSSSEQ ID NO:61CD5-Linker1 nucleic acid sequence

GGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCC

SEQ ID NO:62CD5-Linker1 protein sequence

GGGGSGGGGSGGGGS

SEQ ID NO:63 CD5-CD8a nucleic acid sequences

TTTGTGCCTGTATTTCTGCCTGCCAAGCCCACCACAACACCTGCCCCTAGACCACCCACCCCTGCCCCCACCATTGCTTCTCAGCCCCTTAGCTTAAGACCTGAAGCCTGTAGACCTGCTGCTGGGGGGGCTGTGCACACAAGAGGCCTGGACTTTGCCTGTACTACTACCCCTGCACCTAGGCCTCCCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCTGGCACCTGTGGGGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCACAGAAAC

SEQ ID NO:64 CD5-CD8a protein sequence

FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN

SEQ ID NO:65 CD5-CD28 intracellular domain (IC) nucleic acid sequences

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

SEQ ID NO:66 CD5-CD28 intracellular domain (IC) protein sequences

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

SEQ ID NO:67 CD5-CD3z intracellular signaling domain nucleic acid sequences

AGAGTGAAGTTCAGCAGATCTGCTGATGCCCCTGCCTATCAGCAAGGGCAGAATCAGCTGTACAATGAGCTGAATCTGGGCAGAAGAGAGGAGTATGATGTGCTGGACAAGAGAAGAGGCAGAGACCCTGAGATGGGGGGCAAGCCTAGAAGAAAGAACCCCCAAGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCTGAGGCCTACTCTGAGATTGGCATGAAGGGGGAGAGAAGAAGAGGCAAGGGCCATGATGGCCTGTACCAAGGCCTGAGCACAGCCACCAAGGACACCTATGATGCCCTACACATGCAAGCTCTGCCTCCTAGA

SEQ ID NO:68 CD5-CD3z intracellular signaling domain protein sequences

SEQ ID NO:69 CD5-T2A nucleic acid sequences

GAAGGAAGGGGCAGCCTACTGACCTGTGGGGATGTGGAGGAGAACCCTGGCCCC

SEQ ID NO:70 CD5-T2A protein sequence

EGRGSLLTCGDVEENPGP

SEQ ID NO:71 CD5-CSF2RA signal nucleic acid sequence

ATGTTGCTATTAGTAACCAGCCTGCTGCTGTGTGAGCTGCCCCACCCTGCCTTCCTGTTAATCCCA

SEQ ID NO:72 CD5-CSF2RA signal protein sequence

MLLLVTSLLLCELPHPAFLLIP

SEQ ID NO:73 CD5-tEGFR nucleic acid sequence

CGAAAGGTATGTAATGGCATTGGCATTGGGGAGTTTAAGGACAGCCTGAGCATCAATGCCACCAACATCAAGCACTTCAAGAACTGCACAAGCATCAGTGGGGACTTGCACATCCTGCCTGTGGCCTTCAGAGGGGACAGCTTCACCCACACCCCCCCCCTGGACCCCCAAGAGCTGGACATCCTGAAGACAGTGAAGGAGATCACTGGCTTCTTGCTGATCCAAGCCTGGCCTGAGAACAGAACAGACCTGCATGCCTTTGAGAACCTGGAGATCATCAGAGGCAGAACCAAGCAGCATGGGCAGTTCAGCCTGGCTGTGGTGAGCCTGAACATCACAAGCCTGGGCCTGAGAAGCTTAAAGGAGATCTCTGATGGGGATGTGATCATCTCTGGCAACAAGAACCTGTGCTATGCCAACACCATCAACTGGAAGAAGCTGTTTGGCACCTCTGGGCAGAAGACCAAGATCATCAGCAACAGAGGGGAGAACTCCTGTAAGGCCACTGGCCAAGTGTGTCATGCCCTATGCAGCCCTGAGGGGTGCTGGGGCCCTGAGCCTAGAGACTGTGTGAGCTGCAGAAATGTGAGCAGAGGCAGAGAGTGTGTGGACAAGTGCAACCTGCTGGAGGGGGAGCCTAGAGAGTTTGTGGAGAACTCTGAGTGTATTCAGTGTCATCCTGAGTGCCTGCCCCAAGCCATGAACATCACCTGCACTGGCAGAGGCCCTGACAACTGCATTCAGTGTGCCCACTACATTGATGGCCCCCACTGTGTGAAGACCTGCCCTGCTGGGGTGATGGGGGAGAACAACACCCTGGTGTGGAAGTATGCTGATGCTGGCCATGTGTGTCACCTGTGCCATCCCAACTGCACCTATGGCTGCACTGGCCCTGGCCTGGAGGGCTGCCCCACCAATGGTCCCAAGATTCCTAGCATTGCCACTGGCATGGTGGGGGCCCTGCTCCTACTTCTGGTGGTTGCCCTGGGCATTGGCCTGTTCATG

SEQ ID NO:74CD5-tEGFR protein sequence

RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDI

LKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLK

EISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPE

GCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNIT

CTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCT

YGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

SEQ ID NO:75CD5-VHH-CD28z-CAR nucleic acid sequence

ATGGCCCTACCTGTGACAGCCCTACTGTTACCCCTGGCCCTCCTTCTGCATGCTGCT

AGACCTCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCC

TCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAACTATGCTATCAG

CTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCC

TACAATGGTGACACAAAATATGCACAGAGGCTCCAGGGCAGAGTCACCATGACCA

CAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAACCTAAGATCTGACGA

CACGGCCGTGTATTACTGTGCGCGCTACGAATCTATGTCTGGTCAGGATATCTGGGG

TCAAGGTACTCTGGTGACCGTCTCCTCAGGGGGGGGGGGCTCTGGGGGGGGTGGC

TCAGGTGGCGGTGGCTCTGAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTC

AGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTAGCCAT

AGCGCCATGGGTTGGGTTCGTCAGGCACCTGGTAAAGGTCTGGAATGGGTTAGCA

GCATCTATGCCCGCGGCGGCTATACCTATTATGCAGATAGCGTTAAAGGTCGTTTTA

CCATTAGCCGTGATAACAGCAAAAATACCCTGTACCTGCAGATGAATAGTCTGCGT

GCAGAGGATACCGCAGTGTATTATTGTGCGCGCGGTTACCATCTGGAATACATGGTT

TCTCAGGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCATTTGTGCCTGTA

TTTCTGCCTGCCAAGCCCACCACAACACCTGCCCCTAGACCACCCACCCCTGCCCC

CACCATTGCTTCTCAGCCCCTTAGCTTAAGACCTGAAGCCTGTAGACCTGCTGCTG

GGGGGGCTGTGCACACAAGAGGCCTGGACTTTGCCTGTGACATCTACATCTGGGC

CCCCCTGGCTGGCACCTGTGGGGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACT

GCAACCACAGAAACAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGA

ACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA

CCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGATCTGCTGATGC

CCCTGCCTATCAGCAAGGGCAGAATCAGCTGTACAATGAGCTGAATCTGGGCAGA

AGAGAGGAGTATGATGTGCTGGACAAGAGAAGAGGCAGAGACCCTGAGATGGGG

GGCAAGCCTAGAAGAAAGAACCCCCAAGAGGGCCTGTATAATGAGCTGCAGAAG

GACAAGATGGCTGAGGCCTACTCTGAGATTGGCATGAAGGGGGAGAGAAGAAGA

GGCAAGGGCCATGATGGCCTGTACCAAGGCCTGAGCACAGCCACCAAGGACACCT

ATGATGCCCTACACATGCAAGCTCTGCCTCCTAGAGGCTCTGGGGAAGGAAGGGG

CAGCCTACTGACCTGTGGGGATGTGGAGGAGAACCCTGGCCCCATGTTGCTATTAG

TAACCAGCCTGCTGCTGTGTGAGCTGCCCCACCCTGCCTTCCTGTTAATCCCACGA

AAGGTATGTAATGGCATTGGCATTGGGGAGTTTAAGGACAGCCTGAGCATCAATGC

CACCAACATCAAGCACTTCAAGAACTGCACAAGCATCAGTGGGGACTTGCACATC

CTGCCTGTGGCCTTCAGAGGGGACAGCTTCACCCACACCCCCCCCCTGGACCCCC

AAGAGCTGGACATCCTGAAGACAGTGAAGGAGATCACTGGCTTCTTGCTGATCCA

AGCCTGGCCTGAGAACAGAACAGACCTGCATGCCTTTGAGAACCTGGAGATCATC

AGAGGCAGAACCAAGCAGCATGGGCAGTTCAGCCTGGCTGTGGTGAGCCTGAAC

ATCACAAGCCTGGGCCTGAGAAGCTTAAAGGAGATCTCTGATGGGGATGTGATCAT

CTCTGGCAACAAGAACCTGTGCTATGCCAACACCATCAACTGGAAGAAGCTGTTT

GGCACCTCTGGGCAGAAGACCAAGATCATCAGCAACAGAGGGGAGAACTCCTGT

AAGGCCACTGGCCAAGTGTGTCATGCCCTATGCAGCCCTGAGGGGTGCTGGGGCC

CTGAGCCTAGAGACTGTGTGAGCTGCAGAAATGTGAGCAGAGGCAGAGAGTGTG

TGGACAAGTGCAACCTGCTGGAGGGGGAGCCTAGAGAGTTTGTGGAGAACTCTG

AGTGTATTCAGTGTCATCCTGAGTGCCTGCCCCAAGCCATGAACATCACCTGCACT

GGCAGAGGCCCTGACAACTGCATTCAGTGTGCCCACTACATTGATGGCCCCCACTG

TGTGAAGACCTGCCCTGCTGGGGTGATGGGGGAGAACAACACCCTGGTGTGGAA

GTATGCTGATGCTGGCCATGTGTGTCACCTGTGCCATCCCAACTGCACCTATGGCTG

CACTGGCCCTGGCCTGGAGGGCTGCCCCACCAATGGTCCCAAGATTCCTAGCATTG

CCACTGGCATGGTGGGGGCCCTGCTCCTACTTCTGGTGGTTGCCCTGGGCATTGGC

CTGTTCATGTGA

SEQ ID NO:76CD5-VHH-CD28z-CAR protein sequence

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISW

VRQAPGQGLEWMGWISAYNGDTKYAQRLQGRVTMTTDTSTSTAYMELRNLRSDDTA

VYYCARYESMSGQDIWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPG

GSLRLSCAASGFTFSHSAMGWVRQAPGKGLEWVSSIYARGGYTYYADSVKGRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCARGYHLEYMVSQDVWGQGTLVTVSSFVPVFLP

AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG

VLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR

VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG

LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG

SGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSI

NATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPE

NRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYA

NTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNV

SRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID

GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM*

Here represents protein termination

SEQ ID NO:77BCMA-CAR nucleic acid sequences

tacccatacgatgttccagattacgctgacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatca

cttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcat

ccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaa

gattttgcaacttactactgtcagcaaaaatacgacctcctcacttttggcggagggaccaaggttgagatcaaaggcagcaccagcgg

ctccggcaagcctggctctggcgagggcagcacaaagggacagctgcagctgcaggagtcgggcccaggactggtgaagccttcg

gagaccctgtccctcacctgcactgtctctggtggctccatcagcagtagtagttactactggggctggatccgccagcccccagggaa

ggggctggagtggattgggagtatctcctatagtgggagcacctactacaacccgtccctcaagagtcgagtcaccatatccgtagaca

cgtccaagaaccagttctccctgaagctgagttctgtgaccgccgcagacacggcggtgtactactgcgccagagatcgtggagaca

ccatactagacgtatggggtcagggtacaatggtcaccgtcagctcattcgtgcccgtgttcctgcccgccaaacctaccaccacccct

gcccctagacctcccaccccagccccaacaatcgccagccagcctctgtctctgcggcccgaagcctgtagacctgctgccggcgga

gccgtgcacaccagaggcctggacttcgcctgcgacatctacatctgggcccctctggccggcacctgtggcgtgctgctgctgagcc

tggtgatcaccctgtactgcaaccaccggaacagaagcaagcggagccggctgctgcacagcgactacatgaacatgaccccaaga

cggcctggccccacccggaagcactaccagccttacgcccctcccagagacttcgccgcctaccggtccagagtgaagttcagcag

atccgccgacgcccctgcctaccagcagggacagaaccagctgtacaacgagctgaacctgggcagacgggaagagtacgacgtg

ctggacaagcggagaggccgggaccccgagatgggcggaaagcccagacggaagaacccccaggaaggcctgtataacgaact

gcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcgcggcaagggccacgatggcctgt

accagggcctgagcaccgccaccaaggacacctacgacgccctgcacatgcaggccctgccccccagaSEQ ID NO:78BCMA-CAR protein sequence

YPYDVPDYADIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY

AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKYDLLTFGGGTKVEIKGS

TSGSGKPGSGEGSTKGQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPP

GKGLEWIGSISYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRGDTILDVWGQGTMVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

Here represents protein termination

SEQ ID NO:79 Amino acid sequence of hIL7

MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

SEQ ID NO:80 Amino acid sequence of CCL19

MALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS

SEQ ID NO:81 Amino acid sequence of IL2RB-CD3z

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAYRHQALPPR

SEQ ID NO:82 Amino acid sequence of IL7RaMut

MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCKKIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTCPTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

Reference is made to:

1Blake,S.,Hughes,T.P.,Mayrhofer,G.&Lyons,A.B.The Src/ABL kinase inhibitor dasatinib(BMS-354825)inhibits function of normal human T-lymphocytes in vitro.Clin Immunol 127,330-339,doi:10.1016/j.clim.2008.02.006(2008).

2Schade,A.E.et al.Dasatinib,a small-molecule protein tyrosine kinase inhibitor,inhibits T-cell activation and proliferation.Blood 111,1366-1377,doi:10.1182/blood-2007-04-084814(2008).

3Weber,E.W.et al.Pharmacologic control of CAR-T cell function using dasatinib.Blood advances 3,711-717,doi:10.1182/bloodadvances.2018028720(2019).

4Mestermann,K.et al.The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells.Science translational medicine 11,doi:10.1126/scitranslmed.aau5907(2019).

5Scheiter,M.et al.Protein Kinase Inhibitors CK59 and CID755673Alter Primary Human NK Cell Effector

Functions.Front Immunol

4,66,doi:10.3389/fimmu.2013.00066(2013).

6Lee,K.C.et al.Lck is a key target of imatinib and dasatinib in T-cell activation.Leukemia 24,896-900,doi:10.1038/leu.2010.11(2010).

7Deguchi,Y.et al.Comparison of imatinib,dasatinib,nilotinib and INNO-406in imatinib-resistant cell lines.Leuk Res 32,980-983,doi:10.1016/j.leukres.2007.11.008(2008).

8Burgess,M.R.,Skaggs,B.J.,Shah,N.P.,Lee,F.Y.&Sawyers,C.L.Comparative analysis of two clinically active BCR-ABL kinase inhibitors reveals the role of conformation-specific binding in resistance.Proc Natl Acad Sci U S A 102,3395-3400,doi:10.1073/pnas.0409770102(2005).

9Bradeen,H.A.et al.Comparison of imatinib mesylate,dasatinib(BMS-354825),and nilotinib(AMN107)in an N-ethyl-N-nitrosourea(ENU)-based mutagenesis screen:high efficacy of drug combinations.Blood 108,2332-2338,doi:10.1182/blood-2006-02-004580(2006).

10Wang,X.et al.Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion.Nat Biotechnol 36,946-949,doi:10.1038/nbt.4198(2018).

11Kluesner,M.G.et al.EditR:A Method to Quantify Base Editing from Sanger Sequencing.CRISPR J 1,239-250,doi:10.1089/crispr.2018.0014(2018).

12Scheiter,M.et al.Protein Kinase Inhibitors CK59 and CID755673Alter Primary Human NK Cell Effector

Functions.Front Immunol

4,66,doi:10.3389/fimmu.2013.00066(2013).

sequence listing

<110> Shanghai Reindeer Biotechnology Co., ltd

<120> universal chimeric antigen receptor T cells and uses thereof

<130> P11079-PI-CN

<160> 82

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA12-21nt target DNA sequence

<400> 1

tctacatcat cactgaatac a 21

<210> 2

<211> 20

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA12-20nt target DNA sequence

<400> 2

ctacatcatc actgaataca 20

<210> 3

<211> 19

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA12-19nt target DNA sequence

<400> 3

tacatcatca ctgaataca 19

<210> 4

<211> 18

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA12-18nt target DNA sequence

<400> 4

acatcatcac tgaataca 18

<210> 5

<211> 21

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA16-21nt target DNA sequence

<400> 5

catcactgaa tacatggaga a 21

<210> 6

<211> 20

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA16-20nt target DNA sequence

<400> 6

atcactgaat acatggagaa 20

<210> 7

<211> 19

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA16-19nt target DNA sequence

<400> 7

tcactgaata catggagaa 19

<210> 8

<211> 18

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA16-18nt target DNA sequence

<400> 8

cactgaatac atggagaa 18

<210> 9

<211> 509

<212> PRT

<213> Artificial

<220>

<223> LCK-T316I mutein sequence

<400> 9

Met Gly Cys Gly Cys Ser Ser His Pro Glu Asp Asp Trp Met Glu Asn

1 5 10 15

Ile Asp Val Cys Glu Asn Cys His Tyr Pro Ile Val Pro Leu Asp Gly

20 25 30

Lys Gly Thr Leu Leu Ile Arg Asn Gly Ser Glu Val Arg Asp Pro Leu

35 40 45

Val Thr Tyr Glu Gly Ser Asn Pro Pro Ala Ser Pro Leu Gln Asp Asn

50 55 60

Leu Val Ile Ala Leu His Ser Tyr Glu Pro Ser His Asp Gly Asp Leu

65 70 75 80

Gly Phe Glu Lys Gly Glu Gln Leu Arg Ile Leu Glu Gln Ser Gly Glu

85 90 95

Trp Trp Lys Ala Gln Ser Leu Thr Thr Gly Gln Glu Gly Phe Ile Pro

100 105 110

Phe Asn Phe Val Ala Lys Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe

115 120 125

Phe Lys Asn Leu Ser Arg Lys Asp Ala Glu Arg Gln Leu Leu Ala Pro

130 135 140

Gly Asn Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala

145 150 155 160

Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Gln Asn Gln Gly Glu

165 170 175

Val Val Lys His Tyr Lys Ile Arg Asn Leu Asp Asn Gly Gly Phe Tyr

180 185 190

Ile Ser Pro Arg Ile Thr Phe Pro Gly Leu His Glu Leu Val Arg His

195 200 205

Tyr Thr Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys

210 215 220

Gln Thr Gln Lys Pro Gln Lys Pro Trp Trp Glu Asp Glu Trp Glu Val

225 230 235 240

Pro Arg Glu Thr Leu Lys Leu Val Glu Arg Leu Gly Ala Gly Gln Phe

245 250 255

Gly Glu Val Trp Met Gly Tyr Tyr Asn Gly His Thr Lys Val Ala Val

260 265 270

Lys Ser Leu Lys Gln Gly Ser Met Ser Pro Asp Ala Phe Leu Ala Glu

275 280 285

Ala Asn Leu Met Lys Gln Leu Gln His Gln Arg Leu Val Arg Leu Tyr

290 295 300

Ala Val Val Thr Gln Glu Pro Ile Tyr Ile Ile Ile Glu Tyr Met Glu

305 310 315 320

Asn Gly Ser Leu Val Asp Phe Leu Lys Thr Pro Ser Gly Ile Lys Leu

325 330 335

Thr Ile Asn Lys Leu Leu Asp Met Ala Ala Gln Ile Ala Glu Gly Met

340 345 350

Ala Phe Ile Glu Glu Arg Asn Tyr Ile His Arg Asp Leu Arg Ala Ala

355 360 365

Asn Ile Leu Val Ser Asp Thr Leu Ser Cys Lys Ile Ala Asp Phe Gly

370 375 380

Leu Ala Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg Glu Gly Ala

385 390 395 400

Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ile Asn Tyr Gly Thr

405 410 415

Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu

420 425 430

Ile Val Thr His Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu

435 440 445

Val Ile Gln Asn Leu Glu Arg Gly Tyr Arg Met Val Arg Pro Asp Asn

450 455 460

Cys Pro Glu Glu Leu Tyr Gln Leu Met Arg Leu Cys Trp Lys Glu Arg

465 470 475 480

Pro Glu Asp Arg Pro Thr Phe Asp Tyr Leu Arg Ser Val Leu Glu Asp

485 490 495

Phe Phe Thr Ala Thr Glu Gly Gln Tyr Gln Pro Gln Pro

500 505

<210> 10

<211> 509

<212> PRT

<213> Artificial

<220>

<223> LCK-T316A mutein sequence

<400> 10

Met Gly Cys Gly Cys Ser Ser His Pro Glu Asp Asp Trp Met Glu Asn

1 5 10 15

Ile Asp Val Cys Glu Asn Cys His Tyr Pro Ile Val Pro Leu Asp Gly

20 25 30

Lys Gly Thr Leu Leu Ile Arg Asn Gly Ser Glu Val Arg Asp Pro Leu

35 40 45

Val Thr Tyr Glu Gly Ser Asn Pro Pro Ala Ser Pro Leu Gln Asp Asn

50 55 60

Leu Val Ile Ala Leu His Ser Tyr Glu Pro Ser His Asp Gly Asp Leu

65 70 75 80

Gly Phe Glu Lys Gly Glu Gln Leu Arg Ile Leu Glu Gln Ser Gly Glu

85 90 95

Trp Trp Lys Ala Gln Ser Leu Thr Thr Gly Gln Glu Gly Phe Ile Pro

100 105 110

Phe Asn Phe Val Ala Lys Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe

115 120 125

Phe Lys Asn Leu Ser Arg Lys Asp Ala Glu Arg Gln Leu Leu Ala Pro

130 135 140

Gly Asn Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala

145 150 155 160

Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Gln Asn Gln Gly Glu

165 170 175

Val Val Lys His Tyr Lys Ile Arg Asn Leu Asp Asn Gly Gly Phe Tyr

180 185 190

Ile Ser Pro Arg Ile Thr Phe Pro Gly Leu His Glu Leu Val Arg His

195 200 205

Tyr Thr Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys

210 215 220

Gln Thr Gln Lys Pro Gln Lys Pro Trp Trp Glu Asp Glu Trp Glu Val

225 230 235 240

Pro Arg Glu Thr Leu Lys Leu Val Glu Arg Leu Gly Ala Gly Gln Phe

245 250 255

Gly Glu Val Trp Met Gly Tyr Tyr Asn Gly His Thr Lys Val Ala Val

260 265 270

Lys Ser Leu Lys Gln Gly Ser Met Ser Pro Asp Ala Phe Leu Ala Glu

275 280 285

Ala Asn Leu Met Lys Gln Leu Gln His Gln Arg Leu Val Arg Leu Tyr

290 295 300

Ala Val Val Thr Gln Glu Pro Ile Tyr Ile Ile Ala Glu Tyr Met Glu

305 310 315 320

Asn Gly Ser Leu Val Asp Phe Leu Lys Thr Pro Ser Gly Ile Lys Leu

325 330 335

Thr Ile Asn Lys Leu Leu Asp Met Ala Ala Gln Ile Ala Glu Gly Met

340 345 350

Ala Phe Ile Glu Glu Arg Asn Tyr Ile His Arg Asp Leu Arg Ala Ala

355 360 365

Asn Ile Leu Val Ser Asp Thr Leu Ser Cys Lys Ile Ala Asp Phe Gly

370 375 380

Leu Ala Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg Glu Gly Ala

385 390 395 400

Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ile Asn Tyr Gly Thr

405 410 415

Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu

420 425 430

Ile Val Thr His Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu

435 440 445

Val Ile Gln Asn Leu Glu Arg Gly Tyr Arg Met Val Arg Pro Asp Asn

450 455 460

Cys Pro Glu Glu Leu Tyr Gln Leu Met Arg Leu Cys Trp Lys Glu Arg

465 470 475 480

Pro Glu Asp Arg Pro Thr Phe Asp Tyr Leu Arg Ser Val Leu Glu Asp

485 490 495

Phe Phe Thr Ala Thr Glu Gly Gln Tyr Gln Pro Gln Pro

500 505

<210> 11

<211> 509

<212> PRT

<213> Artificial

<220>

<223> LCK-T316M mutein sequence

<400> 11

Met Gly Cys Gly Cys Ser Ser His Pro Glu Asp Asp Trp Met Glu Asn

1 5 10 15

Ile Asp Val Cys Glu Asn Cys His Tyr Pro Ile Val Pro Leu Asp Gly

20 25 30

Lys Gly Thr Leu Leu Ile Arg Asn Gly Ser Glu Val Arg Asp Pro Leu

35 40 45

Val Thr Tyr Glu Gly Ser Asn Pro Pro Ala Ser Pro Leu Gln Asp Asn

50 55 60

Leu Val Ile Ala Leu His Ser Tyr Glu Pro Ser His Asp Gly Asp Leu

65 70 75 80

Gly Phe Glu Lys Gly Glu Gln Leu Arg Ile Leu Glu Gln Ser Gly Glu

85 90 95

Trp Trp Lys Ala Gln Ser Leu Thr Thr Gly Gln Glu Gly Phe Ile Pro

100 105 110

Phe Asn Phe Val Ala Lys Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe

115 120 125

Phe Lys Asn Leu Ser Arg Lys Asp Ala Glu Arg Gln Leu Leu Ala Pro

130 135 140

Gly Asn Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala

145 150 155 160

Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Gln Asn Gln Gly Glu

165 170 175

Val Val Lys His Tyr Lys Ile Arg Asn Leu Asp Asn Gly Gly Phe Tyr

180 185 190

Ile Ser Pro Arg Ile Thr Phe Pro Gly Leu His Glu Leu Val Arg His

195 200 205

Tyr Thr Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys

210 215 220

Gln Thr Gln Lys Pro Gln Lys Pro Trp Trp Glu Asp Glu Trp Glu Val

225 230 235 240

Pro Arg Glu Thr Leu Lys Leu Val Glu Arg Leu Gly Ala Gly Gln Phe

245 250 255

Gly Glu Val Trp Met Gly Tyr Tyr Asn Gly His Thr Lys Val Ala Val

260 265 270

Lys Ser Leu Lys Gln Gly Ser Met Ser Pro Asp Ala Phe Leu Ala Glu

275 280 285

Ala Asn Leu Met Lys Gln Leu Gln His Gln Arg Leu Val Arg Leu Tyr

290 295 300

Ala Val Val Thr Gln Glu Pro Ile Tyr Ile Ile Met Glu Tyr Met Glu

305 310 315 320

Asn Gly Ser Leu Val Asp Phe Leu Lys Thr Pro Ser Gly Ile Lys Leu

325 330 335

Thr Ile Asn Lys Leu Leu Asp Met Ala Ala Gln Ile Ala Glu Gly Met

340 345 350

Ala Phe Ile Glu Glu Arg Asn Tyr Ile His Arg Asp Leu Arg Ala Ala

355 360 365

Asn Ile Leu Val Ser Asp Thr Leu Ser Cys Lys Ile Ala Asp Phe Gly

370 375 380

Leu Ala Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg Glu Gly Ala

385 390 395 400

Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ile Asn Tyr Gly Thr

405 410 415

Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu

420 425 430

Ile Val Thr His Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu

435 440 445

Val Ile Gln Asn Leu Glu Arg Gly Tyr Arg Met Val Arg Pro Asp Asn

450 455 460

Cys Pro Glu Glu Leu Tyr Gln Leu Met Arg Leu Cys Trp Lys Glu Arg

465 470 475 480

Pro Glu Asp Arg Pro Thr Phe Asp Tyr Leu Arg Ser Val Leu Glu Asp

485 490 495

Phe Phe Thr Ala Thr Glu Gly Gln Tyr Gln Pro Gln Pro

500 505

<210> 12

<211> 22

<212> DNA

<213> Artificial

<220>

<223> LCK-saCas9-sgRNA1 target DNA sequence

<400> 12

ctacatcatc actgaataca tg 22

<210> 13

<211> 22

<212> DNA

<213> Artificial

<220>

<223> LCK-saCas9-sgRNA2 target DNA sequence

<400> 13

atcactgaat acatggagaa tg 22

<210> 14

<211> 20

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA1 target DNA sequence

<400> 14

gctacccgag tcggctacca 20

<210> 15

<211> 20

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA11 target DNA sequence

<400> 15

gctctacgct gtggtcaccc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA8 target DNA sequence

<400> 16

gtattcagtg atgatgtaga 20

<210> 17

<211> 20

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA9 target DNA sequence

<400> 17

tattcagtga tgatgtagat 20

<210> 18

<211> 20

<212> DNA

<213> Artificial

<220>

<223> LCK-sgRNA12 target DNA sequence

<400> 18

ctacatcatc actgaataca 20

<210> 19

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316I-for-sgRNA8 as repair template

<400> 19

acgccttcct ggccgaggcc aacctcatga agcagctgca acaccagcgg ctggttcggc 60

tctacgctgt ggtcacccag gagcccatct acatcatcat tgaatacatg gagaatggtg 120

ggtgctaccc gagtc 135

<210> 20

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316I-for-sgRNA9 as repair template

<400> 20

gacgccttcc tggccgaggc caacctcatg aagcagctgc aacaccagcg gctggttcgg 60

ctctacgctg tggtcaccca ggagcccatc tacatcatca ttgaatacat ggagaatggt 120

gggtgctacc cgagt 135

<210> 21

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316I-for-sgRNA12 as repair template

<400> 21

aaaaggtgta tttccaccct ctggcaggga cagcagggag agcagtatcc cctggtagcc 60

gactcgggta gcacccacca ttctccatgt attcaatgat gatgtagatg ggctcctggg 120

tgaccacagc gtaga 135

<210> 22

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316M-for-sgRNA8 as repair template

<400> 22

acgccttcct ggccgaggcc aacctcatga agcagctgca acaccagcgg ctggttcggc 60

tctacgctgt ggtcacccag gagcccatct acatcatcat ggaatacatg gagaatggtg 120

ggtgctaccc gagtc 135

<210> 23

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316M-for-sgRNA9 as repair template

<400> 23

gacgccttcc tggccgaggc caacctcatg aagcagctgc aacaccagcg gctggttcgg 60

ctctacgctg tggtcaccca ggagcccatc tacatcatca tggaatacat ggagaatggt 120

gggtgctacc cgagt 135

<210> 24

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316M-for-sgRNA12 as repair template

<400> 24

aaaaggtgta tttccaccct ctggcaggga cagcagggag agcagtatcc cctggtagcc 60

gactcgggta gcacccacca ttctccatgt attccatgat gatgtagatg ggctcctggg 120

tgaccacagc gtaga 135

<210> 25

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316A-for-sgRNA8 as repair template

<400> 25

acgccttcct ggccgaggcc aacctcatga agcagctgca acaccagcgg ctggttcggc 60

tctacgctgt ggtcacccag gagcccatct acatcatcgc cgaatacatg gagaatggtg 120

ggtgctaccc gagtc 135

<210> 26

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316A-for-sgRNA9 as repair template

<400> 26

gacgccttcc tggccgaggc caacctcatg aagcagctgc aacaccagcg gctggttcgg 60

ctctacgctg tggtcaccca ggagcccatc tacatcatcg ccgaatacat ggagaatggt 120

gggtgctacc cgagt 135

<210> 27

<211> 135

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316A-for-sgRNA12 as repair template

<400> 27

aaaaggtgta tttccaccct ctggcaggga cagcagggag agcagtatcc cctggtagcc 60

gactcgggta gcacccacca ttctccatgt attccgggat gatgtagatg ggctcctggg 120

tgaccacagc gtaga 135

<210> 28

<211> 265

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316I-for-sgRNA1+sgRNA11 as repair template

<400> 28

aggaaagctc aagaaaccct ccttgcttga gctttgggcc agaagaaaag gtgtatttcc 60

accctctggc agggacagca gggagagcag tatcccctgg tagccgactc gggtagcacc 120

caccattctc catgtattca atgatgatgt agatgggctc ctgggtgacc acagcgtaga 180

gccgaaccag ccgctggtgt tgcagctgct tcatgaggtt ggcctcggcc aggaaggcgt 240

ccggggacat gctgccctgc ttcag 265

<210> 29

<211> 265

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316M-for-sgRNA1+sgRNA11 as repair template

<400> 29

aggaaagctc aagaaaccct ccttgcttga gctttgggcc agaagaaaag gtgtatttcc 60

accctctggc agggacagca gggagagcag tatcccctgg tagccgactc gggtagcacc 120

caccattctc catgtattcc atgatgatgt agatgggctc ctgggtgacc acagcgtaga 180

gccgaaccag ccgctggtgt tgcagctgct tcatgaggtt ggcctcggcc aggaaggcgt 240

ccggggacat gctgccctgc ttcag 265

<210> 30

<211> 265

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316A-for-sgRNA1+sgRNA11 as repair template

<400> 30

aggaaagctc aagaaaccct ccttgcttga gctttgggcc agaagaaaag gtgtatttcc 60

accctctggc agggacagca gggagagcag tatcccctgg tagccgactc gggtagcacc 120

caccattctc catgtattcc gggatgatgt agatgggctc ctgggtgacc acagcgtaga 180

gccgaaccag ccgctggtgt tgcagctgct tcatgaggtt ggcctcggcc aggaaggcgt 240

ccggggacat gctgccctgc ttcag 265

<210> 31

<211> 235

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316I-for-sgRNA1+sgRNA12 as repair template

<400> 31

aggaaagctc aagaaaccct ccttgcttga gctttgggcc agaagaaaag gtgtatttcc 60

accctctggc agggacagca gggagagcag tatcccctgg tagccgactc gggtagcacc 120

caccattctc catgtattca atgatgatgt agatgggctc ctgggtgacc acagcgtaga 180

gccgaaccag ccgctggtgt tgcagctgct tcatgaggtt ggcctcggcc aggaa 235

<210> 32

<211> 235

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316M-for-sgRNA1+sgRNA12 as repair template

<400> 32

aggaaagctc aagaaaccct ccttgcttga gctttgggcc agaagaaaag gtgtatttcc 60

accctctggc agggacagca gggagagcag tatcccctgg tagccgactc gggtagcacc 120

caccattctc catgtattcc atgatgatgt agatgggctc ctgggtgacc acagcgtaga 180

gccgaaccag ccgctggtgt tgcagctgct tcatgaggtt ggcctcggcc aggaa 235

<210> 33

<211> 235

<212> DNA

<213> Artificial

<220>

<223> DNA sequence of ssDNA-T316A-for-sgRNA1+sgRNA12 as repair template

<400> 33

aggaaagctc aagaaaccct ccttgcttga gctttgggcc agaagaaaag gtgtatttcc 60

accctctggc agggacagca gggagagcag tatcccctgg tagccgactc gggtagcacc 120

caccattctc catgtattcc gggatgatgt agatgggctc ctgggtgacc acagcgtaga 180

gccgaaccag ccgctggtgt tgcagctgct tcatgaggtt ggcctcggcc aggaa 235

<210> 34

<211> 1690

<212> PRT

<213> Escherichia coli

<400> 34

Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His

1 5 10 15

Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr

20 25 30

Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met

35 40 45

Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys

50 55 60

Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro

65 70 75 80

Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile

85 90 95

Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala

100 105 110

Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg

115 120 125

Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg

130 135 140

Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His

145 150 155 160

Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp

165 170 175

Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala

180 185 190

Ile Leu Gln Asn Gln Gly Asn Ser Gly Ser Glu Thr Pro Gly Thr Ser

195 200 205

Glu Ser Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala

210 215 220

Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys

225 230 235 240

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

245 250 255

Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr

260 265 270

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

275 280 285

Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met

290 295 300

Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu

305 310 315 320

Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile

325 330 335

Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu

340 345 350

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

355 360 365

Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile

370 375 380

Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile

385 390 395 400

Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn

405 410 415

Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys

420 425 430

Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys

435 440 445

Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro

450 455 460

Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu

465 470 475 480

Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile

485 490 495

Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp

500 505 510

Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys

515 520 525

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

530 535 540

Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys

545 550 555 560

Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr

565 570 575

Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro

580 585 590

Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn

595 600 605

Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile

610 615 620

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

625 630 635 640

Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys

645 650 655

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

660 665 670

Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr

675 680 685

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

690 695 700

Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys

705 710 715 720

Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn

725 730 735

Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala

740 745 750

Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys

755 760 765

Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys

770 775 780

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

785 790 795 800

Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys

805 810 815

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

820 825 830

Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu

835 840 845

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

850 855 860

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

865 870 875 880

Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe

885 890 895

Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His

900 905 910

Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser

915 920 925

Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser

930 935 940

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

945 950 955 960

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

965 970 975

Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg

980 985 990

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln

995 1000 1005

Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu

1010 1015 1020

Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val

1025 1030 1035

Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp

1040 1045 1050

His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn

1055 1060 1065

Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn

1070 1075 1080

Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg

1085 1090 1095

Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn

1100 1105 1110

Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala

1115 1120 1125

Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys

1130 1135 1140

His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

1145 1150 1155

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys

1160 1165 1170

Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys

1175 1180 1185

Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu

1190 1195 1200

Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu

1205 1210 1215

Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg

1220 1225 1230

Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala

1235 1240 1245

Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu

1250 1255 1260

Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu

1265 1270 1275

Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp

1280 1285 1290

Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile

1295 1300 1305

Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser

1310 1315 1320

Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys

1325 1330 1335

Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val

1340 1345 1350

Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser

1355 1360 1365

Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met

1370 1375 1380

Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1385 1390 1395

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro

1400 1405 1410

Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu

1415 1420 1425

Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro

1430 1435 1440

Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys

1445 1450 1455

Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val

1460 1465 1470

Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser

1475 1480 1485

Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys

1490 1495 1500

Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu

1505 1510 1515

Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly

1520 1525 1530

Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys

1535 1540 1545

Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His

1550 1555 1560

Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln

1565 1570 1575

Leu Gly Gly Asp Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile

1580 1585 1590

Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu

1595 1600 1605

Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu

1610 1615 1620

Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu

1625 1630 1635

Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp

1640 1645 1650

Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met

1655 1660 1665

Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val His His His

1670 1675 1680

His His His His His His His

1685 1690

<210> 35

<211> 333

<212> DNA

<213> Artificial

<220>

<223> CD19-78 VL nucleic acid sequence

<400> 35

caggctgtgc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacatcgga aataatgctg taagctggta ccagcagctc 120

ccaggaaagg ctcccaaact cctcatctat tatgatgatc tgctcccctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggttgggtg 300

ttcggcggag ggaccaaggt caccgtccta ggt 333

<210> 36

<211> 111

<212> PRT

<213> Artificial

<220>

<223> CD19-78 VL protein sequence

<400> 36

Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Ser Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 37

<211> 363

<212> DNA

<213> Artificial

<220>

<223> CD19-78 VH nucleic acid sequence

<400> 37

gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagctttacc agctactgga tcggctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 180

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gcgcctgtct 300

tactcttggt cttcttggta ctgggatttc tggggtcaag gtactctggt gaccgtctcc 360

tca 363

<210> 38

<211> 121

<212> PRT

<213> Artificial

<220>

<223> CD19-78 VH protein sequence

<400> 38

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Ser Tyr Ser Trp Ser Ser Trp Tyr Trp Asp Phe Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 39

<211> 45

<212> DNA

<213> Artificial

<220>

<223> CD19-Linker1 nucleic acid sequence

<400> 39

ggtggtggtg gtagcggcgg cggcggctct ggtggtggtg gatcc 45

<210> 40

<211> 15

<212> PRT

<213> Artificial

<220>

<223> CD19-Linker1 protein sequence

<400> 40

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 41

<211> 135

<212> DNA

<213> Artificial

<220>

<223> CD19-CD8a range nucleic acid sequence

<400> 41

actactaccc ctgcacctag gcctcccacc ccagccccaa caatcgccag ccagcctctg 60

tctctgcggc ccgaagcctg tagacctgct gccggcggag ccgtgcacac cagaggcctg 120

gacttcgcct gcgac 135

<210> 42

<211> 45

<212> PRT

<213> Artificial

<220>

<223> CD19-CD8a range protein sequence

<400> 42

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 43

<211> 72

<212> DNA

<213> Artificial

<220>

<223> CD19-CD8a TM nucleic acid sequence

<400> 43

atctacatct gggcccctct ggccggcacc tgtggcgtgc tgctgctgag cctggtgatc 60

accctgtact gc 72

<210> 44

<211> 24

<212> PRT

<213> Artificial

<220>

<223> CD19-CD8a TM protein sequence

<400> 44

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 45

<211> 123

<212> DNA

<213> Artificial

<220>

<223> CD19-CD28 intracellular domain (IC) nucleic acid sequences

<400> 45

agaagcaagc ggagccggct gctgcacagc gactacatga acatgacccc aagacggcct 60

ggccccaccc ggaagcacta ccagccttac gcccctccca gagacttcgc cgcctaccgg 120

tcc 123

<210> 46

<211> 41

<212> PRT

<213> Artificial

<220>

<223> CD19-CD28 intracellular domain (IC) protein sequence

<400> 46

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 47

<211> 336

<212> DNA

<213> Artificial

<220>

<223> CD19-CD3z intracellular Signal Domain nucleic acid sequence

<400> 47

agagtgaagt tcagcagatc cgccgacgcc cctgcctacc agcagggaca gaaccagctg 60

tacaacgagc tgaacctggg cagacgggaa gagtacgacg tgctggacaa gcggagaggc 120

cgggaccccg agatgggcgg aaagcccaga cggaagaacc cccaggaagg cctgtataac 180

gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240

aggcgcggca agggccacga tggcctgtac cagggcctga gcaccgccac caaggacacc 300

tacgacgccc tgcacatgca ggccctgccc cccaga 336

<210> 48

<211> 112

<212> PRT

<213> Artificial

<220>

<223> CD19-CD3z intracellular Signal Domain protein sequence

<400> 48

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 49

<211> 54

<212> DNA

<213> Artificial

<220>

<223> CD19-T2A nucleic acid sequence

<400> 49

gagggaaggg gcagcttatt aacatgtggc gatgtggaag agaaccccgg tccc 54

<210> 50

<211> 18

<212> PRT

<213> Artificial

<220>

<223> CD19-T2A protein sequence

<400> 50

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 51

<211> 66

<212> DNA

<213> Artificial

<220>

<223> CD19-CSF2RA signal nucleic acid sequence

<400> 51

atgctgctgc tcgtgacctc tttactgtta tgtgagctgc cccaccccgc ttttttactg 60

atccct 66

<210> 52

<211> 22

<212> PRT

<213> Artificial

<220>

<223> CD19-CSF2RA signal protein sequence

<400> 52

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 53

<211> 1005

<212> DNA

<213> Artificial

<220>

<223> CD19-tEGFR nucleic acid sequence

<400> 53

cgtaaggtgt gtaacggaat cggcattggc gagttcaagg actctttaag catcaacgcc 60

acaaacatca agcacttcaa gaattgtacc tccatcagcg gcgatttaca cattctcccc 120

gtggcttttc gtggcgattc cttcacccac accccccctc tggaccccca agagctggac 180

attttaaaaa ccgtgaagga gatcaccggc tttctgctga tccaagcttg gcccgagaat 240

cgtaccgacc tccacgcctt cgagaattta gagattattc gtggaaggac caagcagcac 300

ggccagttct ctttagccgt cgtgtcttta aacattacca gcctcggttt aaggtcttta 360

aaggagatta gcgacggcga cgtgatcatc tccggcaaca agaacctctg ctacgccaac 420

accatcaact ggaagaagct gttcggaacc agcggccaaa agaccaagat catcagcaat 480

cgtggagaga actcttgtaa ggccactggt caagtttgcc acgccctctg tagccccgaa 540

ggatgttggg gccccgagcc tagggactgt gttagctgca gaaacgtgag cagaggcaga 600

gagtgtgtgg acaaatgcaa tttactggaa ggagagccta gggagttcgt ggagaacagc 660

gaatgtatcc agtgccaccc cgagtgttta cctcaagcca tgaacatcac ttgtaccgga 720

aggggccccg ataactgcat ccaatgcgcc cactacatcg acggacccca ctgcgtgaaa 780

acttgtcccg ccggagtgat gggagagaat aacactttag tgtggaagta cgccgacgct 840

ggccacgtct gccatctgtg ccaccccaac tgtacctacg gctgcactgg tcccggttta 900

gagggatgtc ctaccaacgg ccccaagatc ccctccatcg ccaccggaat ggtgggcgct 960

ctgttattac tgctggtggt ggctctgggc atcggtttat tcatg 1005

<210> 54

<211> 335

<212> PRT

<213> Artificial

<220>

<223> CD19-tEGFR protein sequence

<400> 54

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 55

<211> 2649

<212> DNA

<213> Artificial

<220>

<223> CD19-CD28z-CAR nucleic acid sequence

<400> 55

atggccctgc ctgtgacagc tctgctcctc cctctggccc tgctgctcca tgccgccaga 60

ccccaggctg tgctgactca gccaccctcg gtgtctgaag cccccaggca gagggtcacc 120

atctcctgtt ctggaagcag ctccaacatc ggaaataatg ctgtaagctg gtaccagcag 180

ctcccaggaa aggctcccaa actcctcatc tattatgatg atctgctccc ctcaggggtc 240

tctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 300

cagtctgagg atgaggctga ttattactgt gcagcatggg atgacagcct gaatggttgg 360

gtgttcggcg gagggaccaa ggtcaccgtc ctaggtggtg gtggtggtag cggcggcggc 420

ggctctggtg gtggtggatc cgaggtgcag ctggtgcagt ctggagcaga ggtgaaaaag 480

cccggggagt ctctgaagat ctcctgtaag ggttctggat acagctttac cagctactgg 540

atcggctggg tgcgccagat gcccgggaaa ggcctggagt ggatggggat catctatcct 600

ggtgactctg ataccagata cagcccgtcc ttccaaggcc aggtcaccat ctcagccgac 660

aagtccatca gcaccgccta cctgcagtgg agcagcctga aggcctcgga caccgccatg 720

tattactgtg cgcgcctgtc ttactcttgg tcttcttggt actgggattt ctggggtcaa 780

ggtactctgg tgaccgtctc ctcattcgtg cccgtgttcc tgcccgccaa acctactact 840

acccctgcac ctaggcctcc caccccagcc ccaacaatcg ccagccagcc tctgtctctg 900

cggcccgaag cctgtagacc tgctgccggc ggagccgtgc acaccagagg cctggacttc 960

gcctgcgaca tctacatctg ggcccctctg gccggcacct gtggcgtgct gctgctgagc 1020

ctggtgatca ccctgtactg caaccaccgg aacagaagca agcggagccg gctgctgcac 1080

agcgactaca tgaacatgac cccaagacgg cctggcccca cccggaagca ctaccagcct 1140

tacgcccctc ccagagactt cgccgcctac cggtccagag tgaagttcag cagatccgcc 1200

gacgcccctg cctaccagca gggacagaac cagctgtaca acgagctgaa cctgggcaga 1260

cgggaagagt acgacgtgct ggacaagcgg agaggccggg accccgagat gggcggaaag 1320

cccagacgga agaaccccca ggaaggcctg tataacgaac tgcagaaaga caagatggcc 1380

gaggcctaca gcgagatcgg catgaagggc gagcggaggc gcggcaaggg ccacgatggc 1440

ctgtaccagg gcctgagcac cgccaccaag gacacctacg acgccctgca catgcaggcc 1500

ctgcccccca gaggatccgg agagggaagg ggcagcttat taacatgtgg cgatgtggaa 1560

gagaaccccg gtcccatgct gctgctcgtg acctctttac tgttatgtga gctgccccac 1620

cccgcttttt tactgatccc tcgtaaggtg tgtaacggaa tcggcattgg cgagttcaag 1680

gactctttaa gcatcaacgc cacaaacatc aagcacttca agaattgtac ctccatcagc 1740

ggcgatttac acattctccc cgtggctttt cgtggcgatt ccttcaccca caccccccct 1800

ctggaccccc aagagctgga cattttaaaa accgtgaagg agatcaccgg ctttctgctg 1860

atccaagctt ggcccgagaa tcgtaccgac ctccacgcct tcgagaattt agagattatt 1920

cgtggaagga ccaagcagca cggccagttc tctttagccg tcgtgtcttt aaacattacc 1980

agcctcggtt taaggtcttt aaaggagatt agcgacggcg acgtgatcat ctccggcaac 2040

aagaacctct gctacgccaa caccatcaac tggaagaagc tgttcggaac cagcggccaa 2100

aagaccaaga tcatcagcaa tcgtggagag aactcttgta aggccactgg tcaagtttgc 2160

cacgccctct gtagccccga aggatgttgg ggccccgagc ctagggactg tgttagctgc 2220

agaaacgtga gcagaggcag agagtgtgtg gacaaatgca atttactgga aggagagcct 2280

agggagttcg tggagaacag cgaatgtatc cagtgccacc ccgagtgttt acctcaagcc 2340

atgaacatca cttgtaccgg aaggggcccc gataactgca tccaatgcgc ccactacatc 2400

gacggacccc actgcgtgaa aacttgtccc gccggagtga tgggagagaa taacacttta 2460

gtgtggaagt acgccgacgc tggccacgtc tgccatctgt gccaccccaa ctgtacctac 2520

ggctgcactg gtcccggttt agagggatgt cctaccaacg gccccaagat cccctccatc 2580

gccaccggaa tggtgggcgc tctgttatta ctgctggtgg tggctctggg catcggttta 2640

ttcatgtga 2649

<210> 56

<211> 882

<212> PRT

<213> Artificial

<220>

<223> CD19-CD28z-CAR protein sequence

<400> 56

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser

20 25 30

Glu Ala Pro Arg Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser

35 40 45

Asn Ile Gly Asn Asn Ala Val Ser Trp Tyr Gln Gln Leu Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val

65 70 75 80

Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala

85 90 95

Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala

100 105 110

Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val

115 120 125

Thr Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

145 150 155 160

Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

165 170 175

Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu

180 185 190

Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser

195 200 205

Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

210 215 220

Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met

225 230 235 240

Tyr Tyr Cys Ala Arg Leu Ser Tyr Ser Trp Ser Ser Trp Tyr Trp Asp

245 250 255

Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Phe Val Pro Val

260 265 270

Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

275 280 285

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

290 295 300

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

305 310 315 320

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

325 330 335

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg

340 345 350

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

355 360 365

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

370 375 380

Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala

385 390 395 400

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

405 410 415

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

420 425 430

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

435 440 445

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

450 455 460

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

465 470 475 480

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

485 490 495

His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser

500 505 510

Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu

515 520 525

Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu

530 535 540

Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys

545 550 555 560

Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys

565 570 575

Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly

580 585 590

Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile

595 600 605

Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp

610 615 620

Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile

625 630 635 640

Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser

645 650 655

Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp

660 665 670

Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr

675 680 685

Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile

690 695 700

Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys

705 710 715 720

His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp

725 730 735

Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys

740 745 750

Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu

755 760 765

Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr

770 775 780

Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile

785 790 795 800

Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu

805 810 815

Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His

820 825 830

Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu

835 840 845

Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met

850 855 860

Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu

865 870 875 880

Phe Met

<210> 57

<211> 354

<212> DNA

<213> Artificial

<220>

<223> CD5-Ab20001-61-VH nucleic acid sequence

<400> 57

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc aactatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagcgcct acaatggtga cacaaaatat 180

gcacagaggc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240

atggagctga ggaacctaag atctgacgac acggccgtgt attactgtgc gcgctacgaa 300

tctatgtctg gtcaggatat ctggggtcaa ggtactctgg tgaccgtctc ctca 354

<210> 58

<211> 118

<212> PRT

<213> Artificial

<220>

<223> CD5-Ab20001-61-VH protein sequence

<400> 58

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asn Gly Asp Thr Lys Tyr Ala Gln Arg Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Glu Ser Met Ser Gly Gln Asp Ile Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 59

<211> 363

<212> DNA

<213> Artificial

<220>

<223> CD5-Ab20001-42-VH nucleic acid sequence

<400> 59

gaagttcagc tgctggaaag cggtggtggt ctggttcagc ctggtggtag cctgcgtctg 60

agctgtgcag caagcggttt tacctttagc catagcgcca tgggttgggt tcgtcaggca 120

cctggtaaag gtctggaatg ggttagcagc atctatgccc gcggcggcta tacctattat 180

gcagatagcg ttaaaggtcg ttttaccatt agccgtgata acagcaaaaa taccctgtac 240

ctgcagatga atagtctgcg tgcagaggat accgcagtgt attattgtgc gcgcggttac 300

catctggaat acatggtttc tcaggatgtt tggggtcaag gtactctggt gaccgtctcc 360

tca 363

<210> 60

<211> 121

<212> PRT

<213> Artificial

<220>

<223> CD5-Ab20001-42-VH protein sequence

<400> 60

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Ser

20 25 30

Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Ala Arg Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr His Leu Glu Tyr Met Val Ser Gln Asp Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 61

<211> 45

<212> DNA

<213> Artificial

<220>

<223> CD5-Linker1 nucleic acid sequence

<400> 61

ggtggtggtg gtagcggcgg cggcggctct ggtggtggtg gatcc 45

<210> 62

<211> 15

<212> PRT

<213> Artificial

<220>

<223> CD5-Linker1 protein sequence

<400> 62

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 63

<211> 381

<212> DNA

<213> Artificial

<220>

<223> CD5-CD8a nucleic acid sequence

<400> 63

tttgtgcctg tatttctgcc tgccaagccc accacaacac ctgcccctag accacccacc 60

cctgccccca ccattgcttc tcagcccctt agcttaagac ctgaagcctg tagacctgct 120

gctggggggg ctgtgcacac aagaggcctg gactttgcct gtactactac ccctgcacct 180

aggcctccca ccccagcccc aacaatcgcc agccagcctc tgtctctgcg gcccgaagcc 240

tgtagacctg ctgccggcgg agccgtgcac accagaggcc tggacttcgc ctgcgacatc 300

tacatctggg cccccctggc tggcacctgt ggggtgctgc tgctgagcct ggtgatcacc 360

ctgtactgca accacagaaa c 381

<210> 64

<211> 127

<212> PRT

<213> Artificial

<220>

<223> CD5-CD8a protein sequence

<400> 64

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

50 55 60

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

65 70 75 80

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

85 90 95

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

100 105 110

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn

115 120 125

<210> 65

<211> 123

<212> DNA

<213> Artificial

<220>

<223> CD5-CD28 intracellular domain (IC) nucleic acid sequence

<400> 65

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 66

<211> 41

<212> PRT

<213> Artificial

<220>

<223> CD5-CD28 intracellular domain (IC) protein sequence

<400> 66

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 67

<211> 336

<212> DNA

<213> Artificial

<220>

<223> CD5-CD3z intracellular Signal Domain nucleic acid sequence

<400> 67

agagtgaagt tcagcagatc tgctgatgcc cctgcctatc agcaagggca gaatcagctg 60

tacaatgagc tgaatctggg cagaagagag gagtatgatg tgctggacaa gagaagaggc 120

agagaccctg agatgggggg caagcctaga agaaagaacc cccaagaggg cctgtataat 180

gagctgcaga aggacaagat ggctgaggcc tactctgaga ttggcatgaa gggggagaga 240

agaagaggca agggccatga tggcctgtac caaggcctga gcacagccac caaggacacc 300

tatgatgccc tacacatgca agctctgcct cctaga 336

<210> 68

<211> 112

<212> PRT

<213> Artificial

<220>

<223> CD5-CD3z intracellular Signal Domain protein sequence

<400> 68

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 69

<211> 54

<212> DNA

<213> Artificial

<220>

<223> CD5-T2A nucleic acid sequence

<400> 69

gaaggaaggg gcagcctact gacctgtggg gatgtggagg agaaccctgg cccc 54

<210> 70

<211> 18

<212> PRT

<213> Artificial

<220>

<223> CD5-T2A protein sequence

<400> 70

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 71

<211> 66

<212> DNA

<213> Artificial

<220>

<223> CD5-CSF2RA signal nucleic acid sequence

<400> 71

atgttgctat tagtaaccag cctgctgctg tgtgagctgc cccaccctgc cttcctgtta 60

atccca 66

<210> 72

<211> 22

<212> PRT

<213> Artificial

<220>

<223> CD5-CSF2RA signal protein sequence

<400> 72

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 73

<211> 1005

<212> DNA

<213> Artificial

<220>

<223> CD5-tEGFR nucleic acid sequence

<400> 73

cgaaaggtat gtaatggcat tggcattggg gagtttaagg acagcctgag catcaatgcc 60

accaacatca agcacttcaa gaactgcaca agcatcagtg gggacttgca catcctgcct 120

gtggccttca gaggggacag cttcacccac accccccccc tggaccccca agagctggac 180

atcctgaaga cagtgaagga gatcactggc ttcttgctga tccaagcctg gcctgagaac 240

agaacagacc tgcatgcctt tgagaacctg gagatcatca gaggcagaac caagcagcat 300

gggcagttca gcctggctgt ggtgagcctg aacatcacaa gcctgggcct gagaagctta 360

aaggagatct ctgatgggga tgtgatcatc tctggcaaca agaacctgtg ctatgccaac 420

accatcaact ggaagaagct gtttggcacc tctgggcaga agaccaagat catcagcaac 480

agaggggaga actcctgtaa ggccactggc caagtgtgtc atgccctatg cagccctgag 540

gggtgctggg gccctgagcc tagagactgt gtgagctgca gaaatgtgag cagaggcaga 600

gagtgtgtgg acaagtgcaa cctgctggag ggggagccta gagagtttgt ggagaactct 660

gagtgtattc agtgtcatcc tgagtgcctg ccccaagcca tgaacatcac ctgcactggc 720

agaggccctg acaactgcat tcagtgtgcc cactacattg atggccccca ctgtgtgaag 780

acctgccctg ctggggtgat gggggagaac aacaccctgg tgtggaagta tgctgatgct 840

ggccatgtgt gtcacctgtg ccatcccaac tgcacctatg gctgcactgg ccctggcctg 900

gagggctgcc ccaccaatgg tcccaagatt cctagcattg ccactggcat ggtgggggcc 960

ctgctcctac ttctggtggt tgccctgggc attggcctgt tcatg 1005

<210> 74

<211> 335

<212> PRT

<213> Artificial

<220>

<223> CD5-tEGFR protein sequence

<400> 74

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 75

<211> 2670

<212> DNA

<213> Artificial

<220>

<223> CD5-VHH-CD28z-CAR nucleic acid sequence

<400> 75

atggccctac ctgtgacagc cctactgtta cccctggccc tccttctgca tgctgctaga 60

cctcaggtgc agctggtgca gtctggggct gaggtgaaga agcctgggtc ctcggtgaag 120

gtctcctgca aggcttctgg aggcaccttc agcaactatg ctatcagctg ggtgcgacag 180

gcccctggac aagggcttga gtggatggga tggatcagcg cctacaatgg tgacacaaaa 240

tatgcacaga ggctccaggg cagagtcacc atgaccacag acacatccac gagcacagcc 300

tacatggagc tgaggaacct aagatctgac gacacggccg tgtattactg tgcgcgctac 360

gaatctatgt ctggtcagga tatctggggt caaggtactc tggtgaccgt ctcctcaggg 420

ggggggggct ctgggggggg tggctcaggt ggcggtggct ctgaagttca gctgctggaa 480

agcggtggtg gtctggttca gcctggtggt agcctgcgtc tgagctgtgc agcaagcggt 540

tttaccttta gccatagcgc catgggttgg gttcgtcagg cacctggtaa aggtctggaa 600

tgggttagca gcatctatgc ccgcggcggc tatacctatt atgcagatag cgttaaaggt 660

cgttttacca ttagccgtga taacagcaaa aataccctgt acctgcagat gaatagtctg 720

cgtgcagagg ataccgcagt gtattattgt gcgcgcggtt accatctgga atacatggtt 780

tctcaggatg tttggggtca aggtactctg gtgaccgtct cctcatttgt gcctgtattt 840

ctgcctgcca agcccaccac aacacctgcc cctagaccac ccacccctgc ccccaccatt 900

gcttctcagc cccttagctt aagacctgaa gcctgtagac ctgctgctgg gggggctgtg 960

cacacaagag gcctggactt tgcctgtgac atctacatct gggcccccct ggctggcacc 1020

tgtggggtgc tgctgctgag cctggtgatc accctgtact gcaaccacag aaacaggagt 1080

aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1140

acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1200

gtgaagttca gcagatctgc tgatgcccct gcctatcagc aagggcagaa tcagctgtac 1260

aatgagctga atctgggcag aagagaggag tatgatgtgc tggacaagag aagaggcaga 1320

gaccctgaga tggggggcaa gcctagaaga aagaaccccc aagagggcct gtataatgag 1380

ctgcagaagg acaagatggc tgaggcctac tctgagattg gcatgaaggg ggagagaaga 1440

agaggcaagg gccatgatgg cctgtaccaa ggcctgagca cagccaccaa ggacacctat 1500

gatgccctac acatgcaagc tctgcctcct agaggctctg gggaaggaag gggcagccta 1560

ctgacctgtg gggatgtgga ggagaaccct ggccccatgt tgctattagt aaccagcctg 1620

ctgctgtgtg agctgcccca ccctgccttc ctgttaatcc cacgaaaggt atgtaatggc 1680

attggcattg gggagtttaa ggacagcctg agcatcaatg ccaccaacat caagcacttc 1740

aagaactgca caagcatcag tggggacttg cacatcctgc ctgtggcctt cagaggggac 1800

agcttcaccc acaccccccc cctggacccc caagagctgg acatcctgaa gacagtgaag 1860

gagatcactg gcttcttgct gatccaagcc tggcctgaga acagaacaga cctgcatgcc 1920

tttgagaacc tggagatcat cagaggcaga accaagcagc atgggcagtt cagcctggct 1980

gtggtgagcc tgaacatcac aagcctgggc ctgagaagct taaaggagat ctctgatggg 2040

gatgtgatca tctctggcaa caagaacctg tgctatgcca acaccatcaa ctggaagaag 2100

ctgtttggca cctctgggca gaagaccaag atcatcagca acagagggga gaactcctgt 2160

aaggccactg gccaagtgtg tcatgcccta tgcagccctg aggggtgctg gggccctgag 2220

cctagagact gtgtgagctg cagaaatgtg agcagaggca gagagtgtgt ggacaagtgc 2280

aacctgctgg agggggagcc tagagagttt gtggagaact ctgagtgtat tcagtgtcat 2340

cctgagtgcc tgccccaagc catgaacatc acctgcactg gcagaggccc tgacaactgc 2400

attcagtgtg cccactacat tgatggcccc cactgtgtga agacctgccc tgctggggtg 2460

atgggggaga acaacaccct ggtgtggaag tatgctgatg ctggccatgt gtgtcacctg 2520

tgccatccca actgcaccta tggctgcact ggccctggcc tggagggctg ccccaccaat 2580

ggtcccaaga ttcctagcat tgccactggc atggtggggg ccctgctcct acttctggtg 2640

gttgccctgg gcattggcct gttcatgtga 2670

<210> 76

<211> 889

<212> PRT

<213> Artificial

<220>

<223> CD5-VHH-CD28z-CAR protein sequence

<400> 76

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly

35 40 45

Thr Phe Ser Asn Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln

50 55 60

Gly Leu Glu Trp Met Gly Trp Ile Ser Ala Tyr Asn Gly Asp Thr Lys

65 70 75 80

Tyr Ala Gln Arg Leu Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser

85 90 95

Thr Ser Thr Ala Tyr Met Glu Leu Arg Asn Leu Arg Ser Asp Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Tyr Glu Ser Met Ser Gly Gln Asp Ile

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu

145 150 155 160

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

165 170 175

Ala Ala Ser Gly Phe Thr Phe Ser His Ser Ala Met Gly Trp Val Arg

180 185 190

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Tyr Ala Arg

195 200 205

Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

210 215 220

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

225 230 235 240

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr His Leu

245 250 255

Glu Tyr Met Val Ser Gln Asp Val Trp Gly Gln Gly Thr Leu Val Thr

260 265 270

Val Ser Ser Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr

275 280 285

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

290 295 300

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

305 310 315 320

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

325 330 335

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

340 345 350

Tyr Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser

355 360 365

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

370 375 380

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg

385 390 395 400

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

405 410 415

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

420 425 430

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

435 440 445

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

450 455 460

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

465 470 475 480

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

485 490 495

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly

500 505 510

Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu

515 520 525

Asn Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu

530 535 540

Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly

545 550 555 560

Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn

565 570 575

Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile

580 585 590

Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu

595 600 605

Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly

610 615 620

Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala

625 630 635 640

Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln

645 650 655

Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg

660 665 670

Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys

675 680 685

Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr

690 695 700

Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys

705 710 715 720

Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys

725 730 735

Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg

740 745 750

Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg

755 760 765

Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu

770 775 780

Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys

785 790 795 800

Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys

805 810 815

Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala

820 825 830

Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly

835 840 845

Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile

850 855 860

Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val

865 870 875 880

Val Ala Leu Gly Ile Gly Leu Phe Met

885

<210> 77

<211> 1464

<212> DNA

<213> Artificial

<220>

<223> BCMA-CAR nucleic acid sequence

<400> 77

tacccatacg atgttccaga ttacgctgac atccagatga cccagtctcc atcctccctg 60

tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagag cattagcagc 120

tatttaaatt ggtatcagca gaaaccaggg aaagccccta agctcctgat ctatgctgca 180

tccagtttgc aaagtggggt cccatcaagg ttcagtggca gtggatctgg gacagatttc 240

actctcacca tcagcagtct gcaacctgaa gattttgcaa cttactactg tcagcaaaaa 300

tacgacctcc tcacttttgg cggagggacc aaggttgaga tcaaaggcag caccagcggc 360

tccggcaagc ctggctctgg cgagggcagc acaaagggac agctgcagct gcaggagtcg 420

ggcccaggac tggtgaagcc ttcggagacc ctgtccctca cctgcactgt ctctggtggc 480

tccatcagca gtagtagtta ctactggggc tggatccgcc agcccccagg gaaggggctg 540

gagtggattg ggagtatctc ctatagtggg agcacctact acaacccgtc cctcaagagt 600

cgagtcacca tatccgtaga cacgtccaag aaccagttct ccctgaagct gagttctgtg 660

accgccgcag acacggcggt gtactactgc gccagagatc gtggagacac catactagac 720

gtatggggtc agggtacaat ggtcaccgtc agctcattcg tgcccgtgtt cctgcccgcc 780

aaacctacca ccacccctgc ccctagacct cccaccccag ccccaacaat cgccagccag 840

cctctgtctc tgcggcccga agcctgtaga cctgctgccg gcggagccgt gcacaccaga 900

ggcctggact tcgcctgcga catctacatc tgggcccctc tggccggcac ctgtggcgtg 960

ctgctgctga gcctggtgat caccctgtac tgcaaccacc ggaacagaag caagcggagc 1020

cggctgctgc acagcgacta catgaacatg accccaagac ggcctggccc cacccggaag 1080

cactaccagc cttacgcccc tcccagagac ttcgccgcct accggtccag agtgaagttc 1140

agcagatccg ccgacgcccc tgcctaccag cagggacaga accagctgta caacgagctg 1200

aacctgggca gacgggaaga gtacgacgtg ctggacaagc ggagaggccg ggaccccgag 1260

atgggcggaa agcccagacg gaagaacccc caggaaggcc tgtataacga actgcagaaa 1320

gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcggag gcgcggcaag 1380

ggccacgatg gcctgtacca gggcctgagc accgccacca aggacaccta cgacgccctg 1440

cacatgcagg ccctgccccc caga 1464

<210> 78

<211> 488

<212> PRT

<213> Artificial

<220>

<223> BCMA-CAR protein sequence

<400> 78

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Asp Ile Gln Met Thr Gln Ser

1 5 10 15

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

20 25 30

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys

35 40 45

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln

50 55 60

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

65 70 75 80

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

85 90 95

Cys Gln Gln Lys Tyr Asp Leu Leu Thr Phe Gly Gly Gly Thr Lys Val

100 105 110

Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu

115 120 125

Gly Ser Thr Lys Gly Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu

130 135 140

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly

145 150 155 160

Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro

165 170 175

Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Ser Tyr Ser Gly Ser Thr

180 185 190

Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr

195 200 205

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

210 215 220

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Asp Thr Ile Leu Asp

225 230 235 240

Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Phe Val Pro Val

245 250 255

Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

260 265 270

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

275 280 285

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

290 295 300

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

305 310 315 320

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg

325 330 335

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

340 345 350

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

355 360 365

Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 79

<211> 177

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of hIL7

<400> 79

Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile

1 5 10 15

Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys

20 25 30

Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu

35 40 45

Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe

50 55 60

Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe

65 70 75 80

Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser

85 90 95

Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr

100 105 110

Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala

115 120 125

Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu

130 135 140

Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu

145 150 155 160

Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu

165 170 175

His

<210> 80

<211> 98

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of CCL19

<400> 80

Met Ala Leu Leu Leu Ala Leu Ser Leu Leu Val Leu Trp Thr Ser Pro

1 5 10 15

Ala Pro Thr Leu Ser Gly Thr Asn Asp Ala Glu Asp Cys Cys Leu Ser

20 25 30

Val Thr Gln Lys Pro Ile Pro Gly Tyr Ile Val Arg Asn Phe His Tyr

35 40 45

Leu Leu Ile Lys Asp Gly Cys Arg Val Pro Ala Val Val Phe Thr Thr

50 55 60

Leu Arg Gly Arg Gln Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu

65 70 75 80

Arg Ile Ile Gln Arg Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg

85 90 95

Ser Ser

<210> 81

<211> 206

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of IL2RB-CD3z

<400> 81

Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn

1 5 10 15

Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly

20 25 30

Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe

35 40 45

Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu

50 55 60

Arg Asp Lys Val Thr Gln Leu Leu Pro Leu Asn Thr Asp Ala Tyr Leu

65 70 75 80

Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val Arg Val

85 90 95

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

100 105 110

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

115 120 125

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

130 135 140

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

145 150 155 160

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

165 170 175

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

180 185 190

Asp Thr Tyr Asp Ala Tyr Arg His Gln Ala Leu Pro Pro Arg

195 200 205

<210> 82

<211> 462

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of IL7RaMut

<400> 82

Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln

1 5 10 15

Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp

20 25 30

Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val

35 40 45

Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val

50 55 60

Asn Ile Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val

65 70 75 80

Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr

85 90 95

Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly

100 105 110

Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys

115 120 125

Pro Glu Ala Pro Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn

130 135 140

Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val

145 150 155 160

Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn

165 170 175

Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln

180 185 190

Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile

195 200 205

Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr

210 215 220

Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro

225 230 235 240

Ile Leu Leu Thr Cys Pro Thr Ile Ser Ile Leu Ser Phe Phe Ser Val

245 250 255

Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys

260 265 270

Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His

275 280 285

Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu

290 295 300

Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg

305 310 315 320

Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu

325 330 335

Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys

340 345 350

Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser Phe Gly Arg Asp Ser

355 360 365

Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile

370 375 380

Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly

385 390 395 400

Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser

405 410 415

Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn

420 425 430

Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln

435 440 445

Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

450 455 460

Claims

1. A method of introducing a T316I mutation in the Lck gene of a cell comprising introducing into said cell a CBE base editor;

the method further comprises introducing into the cell an sgRNA, wherein the sgRNA comprises the amino acid sequence of SEQ ID NOs: 5-8.

2. The method of claim 1, wherein the CBE base editor is an A3A-CBE3 fusion protein.

3. The method of claim 1, wherein the cell is a mammalian cell.

4. The method of claim 1, wherein the cell is a stem cell or an immune cell.

5. The method of claim 4, wherein the immune cells are NK cells or T cells.

6. The method of any one of claims 1-5, wherein the cell expresses a Chimeric Antigen Receptor (CAR).

7. A cell expressing a chimeric antigen receptor, wherein the cell has or is induced to have a killing activity and the cell is engineered such that its killing activity is insensitive to an inhibitor of cellular activity, wherein the engineering is an engineering of an LCK protein tyrosine kinase, the engineered LCK protein tyrosine kinase having a T316I mutation.

8. The cell of claim 7, wherein the inhibitor of cellular activity is a T cell activity inhibitor.

9. The cell of claim 7, wherein the engineering is accomplished using a base editor.

10. The cell of claim 9, wherein the base editor is an ABE or CBE base editor.

11. The cell of claim 7, wherein the T316I mutation is obtained by: introducing into said cell a base editor CBE and an sgRNA whose target sequence comprises the nucleotide sequence of SEQ ID NOs: 5-8.

12. The cell of claim 7, wherein the LCK protein in the cell comprises SEQ ID NOs:9, and a polypeptide comprising the amino acid sequence shown in any one of the above figures.

13. The cell of any one of claims 7-12, wherein the cell is a T cell or NK cell.

14. The cell of claim 13, wherein the cell is further engineered to eliminate or attenuate cell killing activity generated via its cell surface TCR.

15. The cell of claim 13, wherein the TCR-associated gene of the cell is knocked out.

16. The cell of claim 13, wherein the TRAC gene of the cell is knocked out.

17. The cell of claim 13, wherein the cell's β2ιη gene is knocked out; and optionally, the CIITA gene of the cell is knocked out.

18. The cell of claim 8, wherein the inhibitor of T cell activity is a protein tyrosine kinase inhibitor.

19. The cell of claim 8, wherein the inhibitor of T cell activity is an LCK protein tyrosine kinase inhibitor.

20. The cell of claim 8, wherein the inhibitor of T cell activity is dasatinib and/or panatinib.

21. Use of the cell of any one of claims 7-20 in the preparation of a universal CAR-T cell.

22. A method of making a CAR cell comprising engineering the CAR cell such that its CAR-mediated killing activity is insensitive to an inhibitor of T cell activity, wherein the engineering is an engineering of an LCK protein tyrosine kinase, the engineered LCK protein tyrosine kinase having a T316I mutation.

23. The method of claim 22, wherein the T316I mutation is obtained by: introducing into the CAR cell a cytosine base editor and a sgRNA comprising the amino acid sequence of SEQ ID NOs: 5-8.

24. The method of claim 22, wherein the LCK protein tyrosine kinase in the CAR cell comprises SEQ ID NOs:9, and a polypeptide comprising the amino acid sequence shown in any one of the above figures.

25. The method of claim 22, wherein the CAR cell is a T cell or an NK cell.

26. The method of claim 25, wherein the CAR cell is further engineered to eliminate or attenuate cell killing activity generated via its cell surface TCR.

27. The method of claim 25, wherein the TCR-related gene of the CAR cell is knocked out.

28. The method of claim 25, wherein the TRAC gene of the CAR cell is knocked out.

29. The method of claim 25, wherein the CAR cell's β2ιη gene is knocked out; and optionally, the CIITA gene of the CAR cell is knocked out.

30. The method of claim 22, wherein the inhibitor of T cell activity is a protein tyrosine kinase inhibitor.

31. The method of claim 22, wherein the inhibitor of T cell activity is an LCK protein tyrosine kinase inhibitor.

32. The method of claim 22, wherein the inhibitor of T cell activity is dasatinib and/or panatinib.

33. The method of claim 25, wherein the T cell or NK cell is contacted with the T cell activity inhibitor during preparation of the CAR cell from the T cell or NK cell.

34. The method of claim 25, wherein the T cell or NK cell is contacted with the inhibitor of T cell activity while the T316I mutation is being performed.

35. The method of claim 34, wherein the concentration of the T cell activity inhibitor is 100nM.

36. The cell of any one of claims 7-20, wherein the intracellular signaling domain of the CAR comprises:

a signaling domain from a CD3z molecule and a co-stimulatory domain from a CD28 molecule; and

2) Optionally, i) hll 7 and CCL19, or ii) IL2RB and IL7Ra variant wherein the hll 7 comprises SEQ ID NO: 79; the CCL19 comprises the amino acid sequence of SEQ ID NO:80, an amino acid sequence shown in seq id no; the IL2RB and the co-stimulatory domain from the CD28 molecule comprise an IL2RB-CD3z peptide fragment, said IL2RB-CD3z peptide fragment comprising the amino acid sequence of SEQ ID NO:81, and a sequence of amino acids shown in seq id no; the IL7Ra variable comprises SEQ ID NO: 82.

37. The cell of claim 36, wherein the signaling domain from a CD3z molecule comprises the amino acid sequence of SEQ ID NO:48, and the co-stimulatory domain from a CD28 molecule comprises the amino acid sequence set forth in SEQ ID NO: 46.

38. The use of claim 21 or the method of any one of claims 22-35, wherein the intracellular signaling domain of the CAR comprises:

39. The use or method of claim 38, wherein the signaling domain from a CD3z molecule comprises the amino acid sequence of SEQ ID NO:48, and the co-stimulatory domain from a CD28 molecule comprises the amino acid sequence set forth in SEQ ID NO: 46.

40. A pharmaceutical kit or pharmaceutical combination comprising a cell according to any one of claims 7-20 and an inhibitor of cellular activity.

41. The pharmaceutical kit or pharmaceutical combination of claim 40, wherein the cell is a T cell.

42. The pharmaceutical kit or pharmaceutical combination of claim 40, wherein the inhibitor of cellular activity is an inhibitor of T-cell activity.

43. The pharmaceutical kit or pharmaceutical combination of claim 40, wherein the inhibitor of cellular activity is a protein tyrosine kinase inhibitor.

44. The pharmaceutical kit or pharmaceutical combination of claim 42, wherein the inhibitor of T cell activity is an LCK protein tyrosine kinase inhibitor.

45. The pharmaceutical kit or pharmaceutical combination of claim 42, wherein the inhibitor of T cell activity is dasatinib and/or panatinib.

46. Use of a cell according to any one of claims 7-20 in combination with an inhibitor of cellular activity in the preparation of an anti-tumour medicament.

47. The use of claim 46, wherein the cells are T cells.

48. The use of claim 46 or 47, wherein the inhibitor of cellular activity is a T-cell activity inhibitor.

49. The use of claim 48, wherein the inhibitor of T cell activity is a protein tyrosine kinase inhibitor.

50. The use of claim 48, wherein the inhibitor of T cell activity is an LCK protein tyrosine kinase inhibitor.

51. The use of claim 48, wherein said T cell activity inhibitor is dasatinib and/or panatinib.